Use of adrenomedullin and anti-adrenomedullin conjugates for diagnosis and / or prognosis of dementia in treatment or prevention of dementia

Abstract

The present invention relates to the use of adrenomedullin and anti-adrenomedullin conjugates for diagnosing and / or predicting dementia in the treatment or prevention of dementia, and to a method for diagnosing dementia or for determining the risk of a subject having no dementia to suffer from dementia, or monitoring treatment of a subject suffering from dementia or monitoring or guiding intervention of a subject suffering from dementia, or monitoring treatment of a subject at risk of dementia or monitoring or guiding prophylactic intervention of a subject at risk of dementia.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201980012542.1, invention name “Application of adrenomedullin and anti-adrenomedullin conjugates for diagnosing and / or predicting dementia in the treatment or prevention of dementia” and application date February 7, 2019. Technical Field

[0002] The present application relates to the field of disease diagnosis, and in particular to the diagnosis, prediction, treatment or prevention of dementia. Background Art

[0003] Dementia is a clinical syndrome characterized by a constellation of signs and symptoms manifested as memory difficulties, language impairment, psychological and psychiatric changes, and impairment in activities of daily living. The different causes (sometimes referred to as subtypes) of the dementia syndrome are: Alzheimer's disease (about 50% of cases), vascular dementia (about 25%), mixed Alzheimer's disease and vascular dementia (included in the above, 25%), dementia with Lewy bodies (15%), and other causes including frontotemporal dementia, focal dementias (e.g., progressive aphasia), subcortical dementias (e.g., Parkinson's disease dementia), and secondary causes of the dementia syndrome (e.g., intracranial lesions) (together about 5%).

[0004] Alzheimer's disease (AD) is the most prevalent form of dementia. As the world's population ages, the frequency of AD is increasing rapidly, and more and more people are at primary risk for this age-related disease. From the 5.3 million U.S. citizens currently affected, the number of victims will rise to 13 million or more by 2050; the total number of affected individuals worldwide will rise to a staggering 100 million ( Alzheimer’s Association,2015 Alzheimer’s disease facts and figures,Alzheimers Dement2015;11:332–84 The key molecular mechanisms and histopathological hallmarks of AD brains involve a dynamic cascade of multiple biochemical events, including pathological amyloidogenic cleavage of amyloid precursor protein (APP), including amyloid-β peptide (Aβ 1-42 ), the production of various different β-amyloid peptide species including dimers, trimers, and oligomers and the subsequent aggregation and deposition of amyloid peptides in plaques, abnormal hyperphosphorylation and aggregation of tau protein, progressive intracellular neurofibrillary degeneration, changes in the innate immune system and inflammation.

[0005] About 5% of patients develop symptoms before the age of 65 and are characterized as having "early-onset Alzheimer's disease" (EOAD). Most of these patients have the sporadic form of the disease, but 10-15% have the inherited form, which is usually inherited in an autosomal dominant manner. Three genes have been proposed to be involved in the development of EOAD: presenilin 1 and 2 and the amyloid precursor protein (APP) gene. Other candidate genes are also being studied. The inherited form tends to begin at age 30 or 40 and has an aggressive course, while sporadic EOAD tends to begin after age 50 and often has a temporal profile similar to "late-onset Alzheimer's disease" (LOAD).

[0006] Mental status tests assess memory, the ability to solve simple problems, and other thinking skills. Such tests can give an overall picture of whether a person is aware of symptoms, knows the date, time, and where he / she is, can remember a small list of words, follow instructions, and do simple calculations. The Mini-Mental State Examination (MMSE) and the Mini-Cognitive Test are two commonly used tests. The MMSE or Folstein test is a 30-point questionnaire that is widely used in clinical and research settings to measure cognitive impairment ( Pangman et al., 2000. Applied Nursing Research 13(4):209–213; Folstein et al., 1975. Journal of Psychiatric Research. 12(3): 189–98 ). During the MMSE, a health care professional asks the patient a series of questions designed to test a variety of everyday mental skills. The maximum MMSE score is 30 points. Scores of 20 to 24 indicate mild dementia, scores of 13 to 20 indicate moderate dementia, and scores less than 12 indicate severe dementia. On average, the MMSE score of a person with Alzheimer's disease decreases by about 2 to 4 points per year. Advantages of the MMSE include that it does not require specialized equipment or training for administration and that it has both validity and reliability for the diagnosis and longitudinal assessment of Alzheimer's disease. During the mini-cognitive test, a person is asked to complete two tasks, remember and then repeat after a few minutes the names of three common objects, and draw a clock face that displays all 12 numbers in the correct position and shows the time specified by the examiner. The results of this short test can help the doctor determine if further evaluation is needed. Other tests are also used, such as the Hodkinson Abbreviated Mental Test score ( Hodkinson 1972. Age and ageing. 1(4): 233–8 ) or GP cognitive function assessments, computerised tests such as CoPs and the Psychological Attributes Profiling System, and longer formal tests for more in-depth analysis of specific deficits.

[0007] Mild cognitive impairment (MCI) is a heterogeneous clinical condition with several potential causes. However, most MCI represents a transitional state between healthy aging and very mild AD ( DeCarli 2003. Lancet Neurol. 2:15–21Thus, studies have shown that MCI subjects tend to progress to clinically probable AD at a rate of approximately 10%-15% per year ( Markesbery 2010. JAlzheimers Dis. 19:221–228 ).

[0008] Alzheimer's disease is usually diagnosed based on patient history, family history, and behavioral observations. The presence of characteristic neurological and neuropsychological features and the absence of optional conditions are supportive. Advanced medical imaging using computed tomography (CT) or magnetic resonance imaging (MRI) and single photon emission computed tomography (SPECT) or positron emission tomography (PET) can be used to help rule out other brain pathologies or dementia subtypes. In addition, it can predict the transition from the prodromal stage (mild cognitive impairment) to Alzheimer's disease. Assessments of intellectual function, including memory tests, can further characterize the state of the disease. Medical organizations have created diagnostic criteria to simplify and standardize the diagnostic process for practicing physicians. After death, when brain material is available and histological examination can be performed, the diagnosis can be confirmed with very high accuracy.

[0009] To date, only symptomatic treatments exist for this disease, all of which attempt to counteract the disturbance in neurotransmitters. Three cholinesterase inhibitors are currently available and have been approved for the treatment of mild to moderate AD. Another treatment option available for moderate to severe AD is memantine, a noncompetitive antagonist of the N-methyl-D-aspartate receptor. Treatments that can halt or at least effectively alter the course of AD, also known as "disease-modifying" drugs, are still under extensive investigation.

[0010] New therapies are urgently needed to treat affected patients and prevent, delay, slow progression, or improve AD symptoms. It is estimated that if the onset of the disease could be delayed by 5 years, the overall incidence of the disease would be reduced by nearly 50%. Symptomatic treatments are drugs aimed at enhancing cognition or controlling neuropsychological symptoms and usually act through neurotransmitter mechanisms; disease-modifying therapies or treatments (DMTs) are agents that prevent, delay, or slow progression and target the underlying pathophysiological mechanisms of AD. Currently, more than 100 agents are in the development pipeline for AD treatment ( Cummings et al., 2017. Alzheimer’s&Dementia: Translational Research&Clinical Interventions 3:367-384 ).

[0011] Dementia with Lewy bodies (DLB) is a type of dementia that worsens over time. Other symptoms may include fluctuations in alertness, visual hallucinations, slowed movements, difficulty walking, and stiffness. DLB is the most common cause of dementia after Alzheimer's disease and vascular dementia. It usually starts after age 50. About 0.1% of people over age 65 are affected. Men appear to be more susceptible than women. The underlying mechanism involves the formation of Lewy bodies, which are made of a protein called alpha-synuclein, in neurons. The diagnosis may be suspected based on symptoms, and blood tests and medical imaging are done to rule out other possible causes. There is currently no cure for DLB. Treatment is supportive and attempts to relieve certain motor and psychological symptoms associated with the disease. Acetylcholinesterase inhibitors such as donepezil may provide some benefit. Some movement problems may improve with the use of levodopa. For a review, see McKeith et al., 2017. Neurology 89: 88-100 .

[0012] Vascular dementia (VaD), also known as multi-infarct dementia (MID) and vascular cognitive impairment (VCI), is a dementia caused by problems with the blood supply to the brain, usually a series of mild strokes, leading to a gradual, worsening cognitive decline. The term refers to a syndrome consisting of a complex interaction of cerebrovascular disease and risk factors, resulting in structural changes in the brain due to strokes and lesions and resulting cognitive changes. Making the diagnosis requires a temporal relationship between stroke and cognitive impairment. Distinguishing between the different dementia syndromes can be challenging due to often overlapping clinical features and related underlying pathologies. In particular, Alzheimer's disease dementia frequently occurs simultaneously with vascular dementia. People with vascular dementia present with progressive cognitive impairment, usually acutely or subacutely, as in mild cognitive impairment, after multiple cerebrovascular events (strokes). For a review, see . Venkat et al., 2015.ExpNeurol272: 97–108 .

[0013] Frontotemporal dementia (FTD) is the clinical manifestation of frontotemporal lobar degeneration, characterized by progressive neuronal loss involving primarily the frontal or temporal lobes, typically with a loss of more than 70% of spindle neurons, while other neuron types remain intact. FTD accounts for 20% of cases of young-onset dementia. Signs and symptoms typically develop in late adulthood, more typically between the ages of 55 and 65, affecting men and women roughly equally. Common signs and symptoms include marked changes in social and personal behavior, apathy, depressed mood, and deficits in both expressive and receptive language. Currently, there is no cure for FTD, but there are treatments that help relieve symptoms. For a review, see Bott et al., 2014. Neurodegener Dis Manag4(6):439– 454 .

[0014] The peptide adrenomedullin (ADM) was first described in an article by Kitamura et al. Kitamura et al.,1993. Biochemicaland Biophysical ResearchCommunications 192(2):553-560 ) was described as a novel hypotensive peptide containing 52 amino acids isolated from human pheochromocytoma. In the same year, a cDNA encoding a precursor peptide containing 185 amino acids and the complete amino acid sequence of the precursor peptide were reported. In particular, the precursor peptide containing a 21 amino acid signal sequence at the N-terminus was called "adrenomedullin precursor peptide" (pre-proADM). pre-proADM contains 185 amino acids (SEQ ID No.: 1). Mature ADM-NH2 is shown in SEQ ID No.: 4, and ADM-Gly is shown in SEQ No. 5.

[0015] The mature adrenomedullin peptide is an amidated peptide (ADM-NH2) comprising 52 amino acids (SEQ ID No: 4) and including amino acids 95 to 146 of pre-proADM formed by proteolytic cleavage. To date, only a few of the peptide fragments formed in the cleavage of the pre-proADM have been characterized more precisely, in particular the physiologically active peptide adrenomedullin (ADM) and the peptide "PAMP" comprising the 20 amino acids (22-41) following the 21 amino acids of the signal peptide of pre-proADM (SEQ ID No.: 2). In addition, for both ADM and PAMP, physiologically active subfragments have been found and studied in more detail. The discovery and characterization of ADM in 1993 triggered intensive research activities and a large number of publications, the results of which have recently been summarized in various review articles, to which reference is made in particular in the context of the present description. Takahashi 2001. Peptides 22:1691; Eto et al., 2001. Peptides 22:1693-1711 and Hinson et al., 2000 EndocrineReviews21(2):138-167 .

[0016] In scientific research to date, it has been found that ADM can be considered a multifunctional regulatory peptide. It is released into the circulation in part in an inactive form extended by glycine ( Kitamura et al., 1998. Biochem.Biophys.Res.C ommun.244(2):551-555 There are also binding proteins that are specific for ADM and may also modulate the effects of ADM ( Pio et al., 2001. TheJournalofBiologicalChemistry276(15): 12292-12300 ).

[0017] The most important physiological effects of ADMs and PAMPs studied to date are those that affect blood pressure. Thus, ADMs are potent vasodilators.

[0018] In addition, it has been found that another physiologically active peptide PAMP formed from pre-proADM mentioned above also exhibits a hypotensive effect, although it seems to have a different mechanism of action from ADM ( Eto et al., 2001. Peptides22:1693-1711; Hinson et al., 2000 EndocrineReviews21(2):138-167; Kuwasako et al., 1997. FEBS Lett 414(1): 105-110; Kuwasaki et al., 1999. Ann. Clin. Biochem. 36: 622-628; Tsuruda et al., 2001. Life Sci.69(2):239-245; Kangawa et al., EP0 622458) .

[0019] Furthermore, it has been found that in a number of pathological conditions, the concentrations of ADM, which can be measured in the circulation and other biological fluids, are significantly higher than those found in healthy controls. Thus, in patients with congestive heart failure, myocardial infarction, renal disease, hypertensive disease, diabetes, in the acute phase of shock, and in sepsis and septic shock, ADM levels are significantly increased, although to varying degrees. In some of these pathological conditions, the concentrations of PAMPs are also increased, but the plasma levels are reduced relative to ADM ( Eto et al., 2001. Peptides 22:1693-1711 ).

[0020] In addition, it is known that abnormally high concentrations of ADM ( Eto et al. 2001.Peptides22: 1693-1711; Hirata et al., 1996.Journal of Clinical Endocrinology and Metabolism81(4):1449-1453; Ehlenz et al., 1997. ExpClinEndocrinolDiabetes105:156- 162; Tomoda et al., 2001. Peptides 22: 1783-1794; Ueda et al., 1999 Am. J. Respir. Crit. Care Med.160:132-136; Wang et al., 2001. Peptides22:1835-1840 The findings correlate with typical hemodynamic changes known to be common phenomena in patients with sepsis and other severe syndromes such as SIRS as a disease process. Wang,Shock1998,10(5):383-384; Wang et al., 1998. Archives of surgery 133(12): 1298-1304 ) and a large number of acute and chronic diseases ( Parlapiano et al., 1999. European Review for Medical and Pharmacological Sciences 3: 53-61; Hinson et al., 2000 Endocrine Reviews 21(2):138-167 ) plays a key role.

[0021] Several methods have been described for measuring circulating ADM levels: directly or indirectly by determining more stable fragments of the homologous precursor peptide. A method describing an assay for measuring circulating mature ADM has recently been published ( Weber et al. 2017.Journal of applied Laboratory Medicine,2(2):222-233 ).

[0022] Other methods for quantifying fragments derived from ADM precursors have been described, such as MR-proADM ( Morgenthaler et al., 2005. Clin Chem 51(10):1823-9 )、PAMP( Washimine et al., 1994.BiochemBiophysRes Commun 202(2):1081-7 ) and CT-proADM( EP 2 111 552 A commercially available homogeneous time-resolved fluorescence immunoassay for measuring MR-proADM in plasma on a fully automated system is available (BRAHMS MR-proADMKRYPTOR; BRAHMS GmbH, Hennigsdorf, Germany) ( Caruhel et al., 2009. Clin Biochem 42(7-8): 725-8 ). Since these peptides are produced from the same precursor in a certain stoichiometric ratio, their plasma levels are correlated to a certain extent.

[0023] The role of MR-proADM in dementia and AD has been explored in several studies. Plasma levels of MR-proADM measured in patients with probable AD are elevated compared with cognitively normal elderly healthy controls ( Buerger et al., 2009. Biological Psychiatry 2009; 65:979–984 ). MR-proADM blood concentration alone showed a classification accuracy of 47% sensitivity at 81% specificity, and the ratio of MR-proADM to another biomarker, CT-proET-1, showed 66% sensitivity at 81% specificity for the detection of AD. In addition, the plasma concentration of MR-proADM had predictive value in the progression from pre-dementia MCI to clinical AD ( Buerger et al., 2010. J Clin Psychiatry 72 (4):556-563 MR-proADM was also measured in a population-based cohort of more than 5000 individuals without prevalent dementia and was shown to be elevated in participants who developed dementia, but did not demonstrate an increased risk after adjustment for traditional risk factors ( Holm et al., 2017. Journal of Internal Medicine 282: 94-101 In patients participating in a longitudinal study of atherosclerosis, MR-proADM levels increased significantly with the progression of deep white matter lesions (DWML) grade ( Kuriyama et al., 2017. Journal of Alzheimer′s Disease 56: 1253-1262 ). In addition, a significant inverse correlation was observed between MR-proADM levels and cognitive test scores.

[0024] Adrenomedullin has been shown to be increased in the frontal cortex from AD patients compared to age-matched controls ( Ferrero et al., 2017. Mol Neurobiol. doi:10.1007 / s12035-017-0700-6, electronic version ahead of print Subreddit ). However, little is known about plasma ADM in patients with dementia, particularly Alzheimer's disease.

[0025] A subcortical vascular dementia model was replicated in mice by placing microcoils bilaterally over the common carotid arteries. Using mice overexpressing circulating ADM, the effects of ADM on cerebral perfusion, cerebrovascular structure, oxidative stress, white matter changes, cognitive function, and brain levels of cAMP, vascular endothelial growth factor, and basic fibroblast growth factor were evaluated. These data suggest that after chronic cerebral hypoperfusion, ADM promotes arteriogenesis and angiogenesis, inhibits oxidative stress, preserves white matter integrity, and prevents cognitive decline. Therefore, ADM may serve as a strategy to address subcortical vascular dementia. ( Maki et al. 2011.Stroke 42:1122-1128 ). Summary of the invention

[0026] The subject matter of the present invention is a method for:

[0027] a) diagnosis of dementia, or

[0028] b) determine the risk of developing dementia in a subject who does not have dementia, or

[0029] c) monitoring the treatment of a person with dementia or monitoring or directing an intervention for a person with dementia, or

[0030] d) monitoring treatment of subjects at risk of developing dementia or monitoring or directing preventive interventions for subjects at risk of developing dementia,

[0031] wherein the level of mature ADM-NH2 shown in SEQ ID No.: 4 in a body fluid sample of the subject is determined, and wherein the level of mature ADM-NH2 is compared with a threshold level,

[0032] And among them

[0033] a) if the level of mature ADM-NH2 shown in SEQ ID No.: 4 is below the threshold level, the subject is diagnosed as having dementia, or wherein

[0034] b) if the level of mature ADM-NH2 shown in SEQ ID No.: 4 is below the threshold level, the subject has an increased risk of developing dementia, or wherein

[0035] c) if the level of mature ADM-NH2 shown in SEQ ID No.:4 increases during the treatment or intervention, the condition of the subject suffering from dementia or at risk of dementia is improving under the treatment or intervention, and / or if the level of mature ADM-NH2 shown in SEQ ID No.:4 increases to above the level threshold, the intervention can continue.

[0036] Another subject matter of the present invention is a method for:

[0037] a) diagnosis of dementia, or

[0038] b) determine the risk of developing dementia in a subject who does not have dementia, or

[0039] c) monitoring treatment of a subject with dementia or monitoring or directing interventions for a subject with dementia, or

[0040] d) monitoring preventive treatment of subjects at risk of developing dementia or monitoring or directing preventive interventions in subjects at risk of developing dementia,

[0041] wherein a marker ratio is determined, the marker ratio may be a ratio of the determined level of mature ADM-NH2 shown in SEQ ID No.: 4 in the subject's body fluid sample to the determined level of adrenomedullin precursor or a fragment thereof (which is not mature ADM-NH2 shown in SEQ ID No.: 4) in the subject's body fluid sample, and wherein the marker ratio is compared with a ratio threshold value,

[0042] And among them

[0043] a) if the marker ratio of mature ADM-NH2 / adrenomedullin precursor or a fragment thereof is below the ratio threshold, the subject is diagnosed as having dementia, or wherein

[0044] b) if the ratio of the marker of mature ADM-NH2 / adrenomedullin precursor or a fragment thereof is below the ratio threshold, the subject has an increased risk of suffering from dementia, or wherein

[0045] c) if the marker ratio increases during the treatment or intervention, the status of the subject suffering from dementia or at risk of dementia is improving under the treatment or intervention, and wherein if the level of the marker ratio increases to above the ratio threshold, the intervention can continue.

[0046] Alternatively, the determined level of mature ADM-NH2 shown in SEQ ID No.:4 in the body fluid sample of the subject and the determined level of adrenomedullin precursor or a fragment thereof (which is not the mature ADM-NH2 shown in SEQ ID No.:4) in the body fluid sample of the subject are combined by a mathematical algorithm, wherein the result of the algorithm is used to diagnose dementia, or to determine the risk of dementia in a subject without dementia, or to monitor the treatment of a subject with dementia, or to monitor or guide intervention for a subject with dementia, or to monitor preventive treatment for a subject at risk of dementia, or to monitor or guide preventive intervention for a subject at risk of dementia. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 ： Figure 1 A typical bio-ADM dose / signal curve and a bio-ADM dose-signal curve in the presence of 100 μg / mL of antibody NT-H are shown.

[0048] Figure 2 ： Figure 2 Shown are bio-ADM concentrations in the MPP cohort and an independent Alzheimer's disease cohort.

[0049] Figure 3 ： Figure 3Shown is a Kaplan-Meier plot of bio-ADM concentrations in the MPP cohort for prediction of Alzheimer's disease.

[0050] Figure 4 ： Figure 4 Shown are box and whisker plots of bio-ADM concentrations in a sub-cohort of the MPP (case-control) cohort for prediction of Alzheimer's disease and in an independent AD cohort.

[0051] Figure 5 ： Figure 5 Shown is a box-and-whisker plot of MR-proADM concentrations in a sub-cohort of the MPP (case-control) cohort for prediction of Alzheimer's disease.

[0052] Figure 6 ： Figure 6 Shown is a box-and-whisker plot of the bio-ADM / MR-proADM ratio in a sub-cohort of the MPP (case-control) cohort for prediction of Alzheimer's disease.

[0053] Figure 7: FIG. 7 shows ROC plots of bio-ADM (A) and the ratio of bio-ADM to MR-proADM (B) in a sub-cohort of the MPP (case-control) cohort for prediction of Alzheimer's disease.

[0054] Figure 8 ： Figure 8 Shown are bio-ADM values ​​in healthy human subjects after administration of NT-H antibody. DETAILED DESCRIPTION

[0055] A surprising finding of the present invention is that the level of mature ADM is significantly reduced in healthy patients who later develop dementia, particularly AD. In addition, it was surprisingly found that if the subject suffers from dementia, particularly Alzheimer's disease dementia, the level of mature ADM is significantly reduced. From the examples, it can be seen that the baseline level of adrenomedullin, particularly the ADM-NH2 shown in SEQ ID No.: 4, predicts the presence of dementia, particularly Alzheimer's disease dementia.

[0056] Furthermore, it has been surprisingly found that if the marker ratio of ADM-NH2 / adrenomedullin precursor or a fragment thereof is below a certain marker level ratio, the subject is diagnosed as suffering from dementia, in particular AD. Furthermore, it has been surprisingly found that if the marker level ratio of ADM-NH2 / adrenomedullin precursor or a fragment thereof is below a certain marker level ratio threshold, the subject has an increased risk of suffering from dementia. Furthermore, it has been surprisingly found that if the marker level ratio increases during the treatment or intervention, the status of the subject suffering from dementia, in particular AD or at risk of suffering from dementia, in particular AD, is improving under treatment or intervention, and wherein if the level of the marker ratio increases to above the ratio threshold, the intervention can continue.

[0057] Alternatively, the determined level of mature ADM-NH2 shown in SEQ ID No.:4 in the body fluid sample of the subject and the determined level of adrenomedullin precursor or a fragment thereof (which is not the mature ADM-NH2 shown in SEQ ID No.:4) in the body fluid sample of the subject are combined by a mathematical formula or algorithm, wherein the result of the formula or algorithm is used to diagnose dementia, or to determine the risk of dementia in a subject without dementia, or to monitor the treatment of a subject with dementia, or to monitor or guide intervention for a subject with dementia, or to monitor preventive treatment for a subject at risk of dementia, or to monitor or guide preventive intervention for a subject at risk of dementia.

[0058] Thus, the surprising discovery of the present invention is that the level of circulating mature ADM (mature ADM-NH2 shown in SEQ ID No.: 4) is reduced in subjects suffering from dementia or at risk of developing dementia. In addition, the level of circulating adrenomedullin precursor or a fragment thereof (which is not mature ADM-NH2 shown in SEQ ID No.: 4) is increased in subjects suffering from dementia or at risk of developing dementia. Mature ADM (mature ADM-NH2 shown in SEQ ID No.: 4) is known to be a hormone responsible for vascular integrity and endothelial function. It is also known that endothelial dysfunction is associated with a number of the most dreaded human diseases such as peripheral vascular disease, stroke, heart disease, diabetes, chronic renal failure, as well as metastasis and dementia. ( Rajendran et al., 2013. Int. J. Biol. Sci. 9(19): 1057-1069 ).

[0059] High levels of adrenomedullin precursor or its fragments (which are not mature ADM-NH2 shown in SEQ ID No.: 4) in the circulation seem to indicate that the body needs to repair the function of the vascular endothelium and needs to support vascular integrity. However, low levels of mature ADM (mature ADM-NH2 shown in SEQ ID No.: 4) indicate that despite the high levels of pro-ADM, the conversion from ADM-Gly to mature ADM (mature ADM-NH2 shown in SEQ ID No.: 4) seems to be disturbed.

[0060] It is known that MR-proADM concentrations are elevated in the circulation of patients with Alzheimer's disease, patients with MCI or subjects who will develop AD ( Buerger et al., 2009. Biological Psychiatry 2009;65:979–984; Buerger et al., 2010. J Clin Psychiatry 72(4):556-563; Holm et al., 2017. Journal of Internal Medicine Medicine 282:94-101 ). This shows that the ADM synthesis pathway is activated. However, no mention is made of the concentration of bioactive ADM. The present invention shows that the concentration of circulating active ADM (bio-ADM) is surprisingly low in patients with AD and in patients who will develop AD (Example 6). In addition, there is increasing evidence that damage to the brain microvasculature is a contributing factor to the pathophysiology of Alzheimer's disease ( Iadecola 2013. The pathobiology of vascular dementia. Neuron 2013;80(4):844–866) Results from histological evaluation and albumin sampling studies suggest that increased permeability of the blood-brain barrier (BBB) ​​may be a key mechanism ( Benarroch2007, Neurovascular unit dysfunction:a vascular component of Alzheimer disease? , Neurology68(20):1730–1732 In a recent study, it was demonstrated that global BBB leakage in patients with early AD was associated with cognitive decline ( Nation et al., 2019. Nature Medicine https: / / doi.org / 10.1038 / s41591-018-0297-y; van de Haar et al. 2016Radiology281(2):527-535 N-terminal anti-ADM antibodies were shown to stabilize adrenomedullin and cause an increase in circulating active ADM ( Geven et al., 2018, Effects of humanized anti-adrenomedullinantibody Adrecizumab(HAM8101)onvascularbarrierfunctionandsurvivalinrodentmodelsof ofadrenomedullinandtheanti-adrenomedullinantibodyAdrecizumabinsepsis,Shock50 (2):132-140 ). In Example 7 and Figure 8 The effect of causing a rapid increase in bio-ADM in the blood of healthy patients is shown in . Increased ADM in the circulation results in beneficial effects on endothelial cells, such as a reduction in capillary leakage. For example, N-terminal anti-ADM antibodies (HAM8101, Adrecizumab) have been shown to enhance endothelial barrier function in experimental models of systemic inflammation and sepsis ( Geven et al., 2018, Effects of humanized anti-adrenomedullinantibodyAdrecizumab (HAM8101)onvascular barrier functionand survivalinrodent modelsofsystemicinf lammationandsepsis, Shock50(6):648-654 ). Therefore, N-terminal ADM binders, more specifically N-terminal anti-ADM antibodies, can be used to increase the concentration of bio-ADM in the blood of patients suffering from or at risk of developing dementia, especially patients suffering from Alzheimer's disease dementia.

[0061] Therefore, in summary, in patients whose bodies need to repair endothelial function and support vascular integrity, low levels of ADM, particularly bioactive ADM, in the circulation may indicate that the processing of ADM is disturbed in the patient. Patients whose bodies need to repair endothelial function and support vascular integrity can be characterized and identified as outlined in the methods of the present invention by determining the level of mature ADM-NH2 shown in SEQ ID No.: 4 in a body fluid sample of the subject and comparing the level of mature ADM-NH2 with a threshold level, or as outlined in the methods of the present invention by determining a marker ratio, which can be the ratio of the level of mature ADM-NH2 shown in SEQ ID No.: 4 in the body fluid sample of the subject to the level of adrenomedullin precursor or a fragment thereof (which is not mature ADM-NH2 shown in SEQ ID No.: 4) in the body fluid sample of the subject, and comparing the marker ratio with a threshold ratio.

[0062] Additionally or alternatively, a patient whose body is in need of repairing endothelial function and in need of supporting vascular integrity may be a patient with global BBB leakage or BBB breakdown. Global BBB leakage or BBB breakdown can be determined as follows: measuring the cerebrospinal fluid (CSF) / serum ratio of albumin or immunoglobulin G (IgG) ( Akaishi et al., 2015. Neurology andClinicalNeuroscience 3:94–100 ), or imaging techniques such as dynamic sensitivity contrast-enhanced magnetic resonance imaging (DSC-MRI) or dynamic contrast-enhanced MRI (DCE-MRI) ( Raja et al., 2018.Neuropharmacology134:259- 271 ).

[0063] Therefore, stratification and identification of patients who require increased bio-ADM levels for the purpose of preventing or arresting the development of cognitive disorders in humans or for the purpose of preventing or treating dementia is performed by any of the methods described above.

[0064] In the examples and Figure 8 It has been shown in that after administration of N-terminal anti-ADM antibodies, bio-ADM in the blood of healthy patients increases rapidly, which may help repair a leaky or damaged blood-brain barrier. Therefore, it seems reasonable that administration of N-terminal anti-ADM antibodies to subjects identified and / or stratified as described above may help prevent and treat dementia.

[0065] Therefore, another object is to provide treatment in subjects having reduced levels of mature ADM (mature ADM-NH2 as shown in SEQ ID No.: 4) and / or a reduced ratio of the level of mature ADM-NH2 as shown in SEQ ID No.: 4 determined in a body fluid sample of said subject to the level of adrenomedullin precursor or a fragment thereof (which is not mature ADM-NH2 as shown in SEQ ID No.: 4) determined in a body fluid sample. Said patient group can be treated with an anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig backbone for the prevention or treatment of dementia in a subject, wherein said anti-ADM antibody or anti-ADM fragment or anti-ADM non-Ig backbone binds to the N-terminal part (aa 1-21) of adrenomedullin:

[0066] YRQSMNNFQGLRSFGCRFGTC (SEQ ID No.: 21).

[0067] In another embodiment of the present invention, the subject to be treated shows signs of mild cognitive impairment or signs of dementia in addition to the above mentioned criteria.

[0068] It is known that administration of anti-adrenomedullin (ADM) antibodies or anti-adrenomedullin antibody fragments or anti-ADM non-Ig backbones to a subject increases the concentration of mature ADM (mature ADM-NH2 shown in SEQ ID No.: 4) in the subject's circulation and thereby improves the condition of a subject suffering from dementia or at risk of dementia.

[0069] The subject matter of the present invention is a method for:

[0070] a) diagnosis of dementia, or

[0071] b) determine the risk of developing dementia in a subject who does not have dementia, or

[0072] c) monitoring treatment of a subject with dementia or monitoring or directing interventions for a subject with dementia, or

[0073] d) monitoring treatment of subjects at risk of developing dementia or monitoring or directing preventive interventions for subjects at risk of developing dementia,

[0074] wherein the level of mature ADM-NH2 shown in SEQ ID No.: 4 in a body fluid sample of the subject is determined, and wherein the level of mature ADM-NH2 is compared with a threshold level,

[0075] And among them

[0076] a) if the level of mature ADM-NH2 shown in SEQ ID No.: 4 is below the threshold level, the subject is diagnosed as having dementia, or wherein

[0077] b) if the level of mature ADM-NH2 shown in SEQ ID No.: 4 is below the threshold level, the subject has an increased risk of developing dementia, or wherein

[0078] c) if the level of mature ADM-NH2 shown in SEQ ID No.:4 increases during the treatment or intervention, the condition of the subject suffering from dementia or at risk of dementia is improving under the treatment or intervention, and / or if the level of mature ADM-NH2 shown in SEQ ID No.:4 increases to above the said level threshold, the intervention can continue.

[0079] In one embodiment, the subject matter of the present invention is a method for:

[0080] a) diagnosis of dementia, or

[0081] b) determine the risk of developing dementia in a subject who does not have dementia, or

[0082] c) monitoring treatment of a subject with dementia or monitoring or directing interventions for a subject with dementia, or

[0083] d) monitoring preventive treatment of subjects at risk of developing dementia or monitoring or directing preventive interventions in subjects at risk of developing dementia,

[0084] wherein a marker ratio is determined, which may be a ratio of a determined level of mature ADM-NH2 as shown in SEQ ID No.: 4 in a body fluid sample of the subject to a determined level of adrenomedullin precursor or a fragment thereof (which is not mature ADM-NH2 as shown in SEQ ID No.: 4) in a body fluid sample of the subject, and wherein the marker ratio is compared with a ratio threshold value,

[0085] And among them

[0086] a) if the marker ratio of mature ADM-NH2 / adrenomedullin precursor or a fragment thereof is below the ratio threshold, the subject is diagnosed as having dementia, or wherein

[0087] b) if the ratio of the marker of mature ADM-NH2 / adrenomedullin precursor or a fragment thereof is below the ratio threshold, the subject has an increased risk of suffering from dementia, or wherein

[0088] c) if the marker ratio increases during the treatment or intervention, the status of the subject suffering from dementia or at risk of dementia is improving under the treatment or intervention, and wherein if the level of the marker ratio increases to above the ratio threshold, the intervention can continue.

[0089] Alternatively, the determined level of mature ADM-NH2 shown in SEQ ID No.:4 in the body fluid sample of the subject and the determined level of adrenomedullin precursor or a fragment thereof (which is not mature ADM-NH2 shown in SEQ ID No.:4) in the body fluid sample of the subject are combined in a mathematical formula or algorithm, wherein the result of the formula or algorithm is used to diagnose dementia, or to determine the risk of dementia in a subject without dementia, or to monitor the treatment of a subject with dementia, or to monitor or guide intervention for a subject with dementia, or to monitor preventive treatment for a subject at risk of dementia, or to monitor or guide preventive intervention for a subject at risk of dementia.

[0090] In one embodiment of the present invention, the levels of two markers are determined in any case: the level of mature ADM-NH2 shown in SEQ ID No.: 4 in a body fluid sample of the subject, and the level of adrenomedullin precursor or a fragment thereof (which is not mature ADM-NH2 shown in SEQ ID No.: 4) in a body fluid sample of the subject. The levels of the two markers are used to perform a calculation, which can be a ratio of the two markers (e.g., a ratio between mature ADM-NH2 and pro-ADM or a fragment thereof or a ratio between proADM or a fragment thereof and mature ADM-NH2), or a mathematical formula in which the two markers are introduced or a mathematical algorithm in which the two markers are introduced. The result of this ratio or mathematical formula or mathematical algorithm can be a value, which is then compared with a predetermined threshold value, and this comparison is then used to diagnose dementia, or to determine the risk of dementia in a subject without dementia, or to monitor the treatment of a subject with dementia or to monitor or guide intervention in a subject with dementia, or to monitor preventive treatment of a subject at risk of dementia or to monitor or guide preventive intervention in a subject at risk of dementia.

[0091] In one embodiment of the subject matter of the present invention, the fragment of adrenomedullin precursor is selected from PAMP (SEQ ID No.: 2), MR-proADM (SEQ ID No.: 3), ADM-Gly (SEQ ID No.: 5) and CT-proADM (SEQ ID No.: 6).

[0092] In one embodiment of the subject matter of the present invention, the threshold level of mature ADM-NH2 shown in SEQ ID No.: 4 is equal to or lower than 15 pg / mL, preferably equal to or lower than 10 pg / mL, preferably equal to or lower than 5 pg / mL.

[0093] In one embodiment of the inventive subject matter, the marker level ratio threshold is in the range of 0.2 to 0.75, preferably 0.3 to 0.6, preferably 0.4 to 0.5.

[0094] In order to calculate the ratio, the concentrations of the two markers must preferably be expressed in the same units (eg pmol / L).

[0095] In one embodiment of the subject matter of the present invention, the body fluid sample is selected from patients with mild cognitive impairment (MCI), Alzheimer's disease, vascular dementia, mixed Alzheimer's disease and vascular dementia, Lewy body dementia, frontotemporal dementia, focal dementia (e.g., progressive aphasia), subcortical dementia (e.g., Parkinson's disease dementia), and secondary causes of dementia syndromes (e.g., intracranial lesions).

[0096] In one embodiment of the present subject matter, the bodily fluid sample is obtained from a subject who has never had a diagnosis of dementia or MCI at the time of sampling.

[0097] In one embodiment of the present subject matter, at least one additional clinical parameter is determined, which is selected from age, race, mental status test (e.g., Mini-Mental State Examination (MMSE)), neuroimaging (CT, MRT, PET, SPECT), family history, ApoE4 genotype, amyloid peptide β1-42 (Aβ 1-42 ), amyloid peptide β1-40 (Aβ 1-40 ), total Tau protein, phosphorylated Tau protein (p-Tau 181, p-Tau 199, p-Tau 231).

[0098] In one embodiment of the present subject matter, the level of said marker is determined by an immunoassay.

[0099] In one embodiment of the present subject matter, the method is used for patient stratification to select patients for treatment with an anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold for preventing or treating dementia in a subject, wherein the anti-ADM antibody or anti-ADM fragment or anti-ADM non-Ig scaffold binds to the N-terminal portion (aa 1-21) of adrenomedullin:

[0100] YRQSMNNFQGLRSFGCRFGTC (SEQ ID No.: 21).

[0101] The subject matter of the present invention is a use of an anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or an anti-ADM non-Ig scaffold for preventing and treating dementia in a subject, wherein the anti-ADM antibody or anti-ADM fragment or anti-ADM non-Ig scaffold binds to the N-terminal portion (aa 1-21) of adrenomedullin:

[0102] YRQSMNNFQGLRSFGCRFGTC (SEQ ID No.: 21).

[0103] The subject matter of the present invention is the use of an anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or an anti-ADM non-Ig skeleton in the prevention and treatment of dementia in a subject, wherein the level of mature ADM-NH2 determined by SEQ ID No.: 4 in the subject's body fluid sample is lower than a threshold level, and / or the subject has a certain marker ratio, which is the ratio of the level of mature ADM-NH2 determined by SEQ ID No.: 4 in the subject's body fluid sample to the level of adrenomedullin precursor or its fragment determined in the subject's body fluid sample, and wherein the marker level ratio is lower than the ratio threshold.

[0104] The subject matter of the present invention is the use of an anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or an anti-ADM non-Ig backbone in preventing and treating dementia in a subject, wherein the fragment of the adrenomedullin precursor is selected from PAMP (SEQ ID No.: 2), MR-proADM (SEQ ID No.: 3), ADM-Gly (SEQ ID No.: 5) and CT-proADM (SEQ ID No.: 6).

[0105] The subject matter of the present invention is the use of an anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or an anti-ADM non-Ig scaffold for preventing and treating dementia in a subject, wherein the subject is selected by the method explained above.

[0106] The subject matter of the present invention is the use of an anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or an anti-ADM non-Ig skeleton in the prevention and treatment of dementia in a subject, wherein the threshold level of mature ADM-NH2 shown in SEQ ID No.: 4 is equal to or lower than 15pg / mL, preferably equal to or lower than 10pg / mL, preferably equal to or lower than 5pg / mL.

[0107] The subject matter of the present invention is the use of an anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or an anti-ADM non-Ig backbone in the prevention and treatment of dementia in a subject, wherein the marker level ratio is in the range of 0.2 to 0.75, preferably 0.3 to 0.6, preferably 0.4 to 0.5.

[0108] The subject matter of the present invention is the use of an anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or an anti-ADM non-Ig skeleton in the prevention and treatment of dementia in a subject, wherein the subject is selected according to the method explained above, and wherein the body fluid sample is selected from blood, serum, plasma, urine, cerebrospinal fluid (CSF) and saliva.

[0109] The subject matter of the present invention is the use of an anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or an anti-ADM non-Ig backbone for the prevention and treatment of dementia in a subject, wherein at least one additional clinical parameter is determined, which is selected from age, race, mental status test (e.g., Mini-Mental State Examination (MMSE)), neuroimaging (CT, MRT, PET, SPECT), family history, ApoE4 genotype, amyloid peptide beta 1-42 (Aβ 1-42 ), amyloid peptide β1-40 (Aβ 1-40 ), total Tau protein, phosphorylated Tau protein (p-Tau 181, p-Tau 199, p-Tau231).

[0110] The subject matter of the present invention is the use of an anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or an anti-ADM non-Ig backbone for preventing and treating dementia in a subject, wherein the level of the marker is determined by immunoassay.

[0111] Mature ADM, bio-ADM and ADM-NH2 are used synonymously throughout this application and are the molecule shown in SEQ ID No.:4.

[0112] As used herein, the term "PAMP" includes both circulating forms of PAMPs, namely, biologically inactive C-terminal glycine extended PAMPs (PAMP-Gly) and biologically active C-terminal amidated PAMPs (PAMP-Amide).

[0113] In a specific embodiment of the present invention, the proADM and / or fragments thereof having at least 5 amino acids and mature ADM are selected from the group consisting of:

[0114] SEQ ID No.: 1 (pre-proADM): amino acids 1-185

[0115] MKLVSVALMYLGSLAFLGADTARLDVASEFRKKWNKWALSRGKRELRMSSSYPTGLADVKAGPAQTLIRPQDMKGASRSPEDSSPDAARIRVKRYRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVAPRSKISPQGYGRRRRRSLPEAGPGRTLVSSKPQAHGAPAPPSGSAPHFL

[0116] SEQ ID No.: 2 (adrenomedullin precursor N-20 terminal peptide, PAMP): amino acids 22-41 of preproADM

[0117] ARLDVASEFRKKWNKWALS R

[0118] SEQ ID No.: 3 (MR-proADM): amino acids 45-92 of preproADM

[0119] ELRMSSSYPTGLADVKAGPAQTLIRPQDMKGASRSPEDSSPDAARIRV

[0120] SEQ ID No.: 4 (mature adrenomedullin (mature ADM); amidated ADM; bio-ADM; hADM): amino acids 95-146 - CONH2

[0121] YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVAPRSKISPQGY-CONH2

[0122] SEQ ID No.: 5 (Adrenomedullin 1-52-Gly (ADM 1-52-Gly)): amino acids 95-147 of preproADM

[0123] YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVAPRSKISPQGYG

[0124] SEQ ID No.: 6 (C-terminal pro-adrenomedullin, CT-proADM): amino acids 148-185 of preproADM

[0125] RRRRRSLPEAGPGRTLVSSKPQAHGAPAPPSGSAPHFL

[0126] In a specific embodiment of the present invention, the immunoreactivity level of mature ADM-NH2 (SEQ ID No.: 4) in the body fluid of said subject is below a threshold value.

[0127] In a specific embodiment of the present invention, the immunoreactivity level of PAMP (SEQ ID No.: 2) or the immunoreactivity level of MR-proADM (SEQ ID No.: 3) or the immunoreactivity level of CT-proADM (SEQ ID No.: 6) or the immunoreactivity level of ADM 1-52-Gly (SEQ ID No.: 5) in the body fluid of the subject is higher than a threshold value.

[0128] In a specific embodiment of the present invention, the ratio of the immunoreactivity level of mature ADM-NH2 (SEQ ID No.: 4) to the immunoreactivity level of MR-proADM (SEQ ID No.: 3) in the body fluid of said subject is below a threshold value.

[0129] In a specific embodiment of the present invention, the level of mature ADM-NH2 is determined using at least one binder selected from the following: a binder that binds to a region contained within the following mature ADM-NH2 sequence (SEQ ID No.: 4), and a second binder that binds to a region contained within the mature ADM-NH2 sequence (SEQ ID NO.: 4).

[0130] In a specific embodiment of the present invention, the level of proADM and / or its fragments is determined using at least one binder selected from the following: a binder that binds to a region contained within the MR-proADM sequence (SEQ ID No.: 3), and a second binder that binds to a region contained within the MR-proADM sequence (SEQ ID No.: 3).

[0131] In a specific embodiment of the present invention, the level of pro-ADM and / or its fragments is determined using at least one binder selected from the following: a binder that binds to a region contained within the CT-proADM sequence (SEQ ID No.: 6), and a second binder that binds to a region contained within the CT-proADM sequence (SEQ ID No.: 6).

[0132] In a specific embodiment of the present invention, the level of pro-ADM and / or its fragments is determined using at least one binder selected from the following: a binder that binds to a region contained within the PAMP sequence (SEQ ID No.: 2), and a second binder that binds to a region contained within the PAMP sequence (SEQ ID No.: 2).

[0133] In a specific embodiment of the present invention, the level of pro-ADM and / or its fragments is determined using at least one binder selected from the following: a binder that binds to a region contained within the ADM 1-52-Gly sequence (SEQ ID No.: 5), and a second binder that binds to a region contained within the ADM 1-52-Gly sequence (SEQ ID No.: 5).

[0134] The subject matter of the present invention is a method according to the invention, wherein the binder is selected from antibodies, antibody fragments or non-Ig scaffolds that bind to pro-adrenomedullin or a fragment thereof having at least 5 amino acids.

[0135] Another embodiment of the present invention relates to a method according to the preceding embodiment, wherein the body fluid can be selected from blood, serum, plasma, urine, cerebrospinal fluid (CSF) and saliva. In a more specific embodiment of the present invention, the body fluid is a blood sample. The blood sample can be selected from whole blood, serum and plasma. In a specific embodiment of the present invention, the sample is selected from human citrate plasma, heparin plasma and EDTA plasma.

[0136] The subject matter of the present invention is a method according to the invention, wherein the determination of the adrenomedullin precursor or a fragment thereof having at least 5 amino acids is performed more than once in one patient.

[0137] The subject of the invention is a method according to the invention, wherein said monitoring is performed in order to assess the response of said subject to the preventive and / or therapeutic measures taken.

[0138] The subject matter of the invention is a method according to the invention, wherein said method is used to stratify said subjects into a plurality of risk groups.

[0139] As used herein, the term "risk" refers to the likelihood of suffering an unwanted event or effect, such as a disease.

[0140] Another embodiment of the present application relates to a method according to the preceding embodiments, wherein a decrease in the level of mature ADM-NH2 predicts an increased risk of developing dementia.

[0141] Another embodiment of the present application relates to a method according to the preceding embodiments, wherein a decrease in the ratio between mature ADM-NH2 and proADM or a fragment thereof selected from MR-proADM, CT-proADM, ADM-Gly and / or PAMP predicts an increased risk of dementia.

[0142] The subject matter of the present invention is also a method for determining the risk of suffering from dementia as defined in any one of the aforementioned paragraphs, wherein the method is carried out so that the object is stratified into the risk group further defined hereinafter. In a specific embodiment of the present invention, the method is used to stratify the object into risk groups, for example, with a low risk, medium risk or high risk group suffering from dementia. The low risk of suffering from dementia means that the value of ripe ADM-NH2 is not substantially reduced compared with the predetermined value in the healthy subjects who do not suffer from dementia. When the level of ripe ADM-NH2 is reduced compared with the predetermined value in the healthy subjects who do not suffer from dementia, there is medium risk, and when the level of ripe ADM-NH2 is significantly reduced when baseline measurement and continues to reduce during subsequent analysis, there is high risk.

[0143] The risk of dementia means the risk of developing dementia within a certain period of time. In a specific embodiment, the period of time is within 10 years or within 7 years or within 5 years or within 2.5 years.

[0144] The term "elevated level" means a level above a certain threshold level.

[0145] The term "reduced level" means a level below a certain threshold level.

[0146] In a specific embodiment of the invention, an assay is used to determine the level of mature ADM-NH2, wherein the assay has an assay sensitivity of <15 pg / mL, preferably <10 pg / mL, more preferably <5 pg / mL.

[0147] In a specific embodiment of the invention, an assay is used to determine the level of MR-proADM, wherein the assay sensitivity of the assay is capable of quantifying MR-proADM in healthy subjects and is <0.5 nmol / L, preferably <0.4 nmol / L, more preferably <0.2 nmol / L.

[0148] In a specific embodiment of the invention, an assay is used to determine the level of CT-proADM, wherein the assay sensitivity of the assay is capable of quantifying CT-proADM in healthy subjects and is <100 pmol / L, preferably <75 pmol / , more preferably <50 pmol / L.

[0149] Another embodiment of the present application relates to a method according to the aforementioned embodiment, wherein an assay is used to determine the level of PAMP-amide, wherein the assay sensitivity of the assay is capable of quantifying PAMP-amide in healthy subjects and is <0.3pmol / L, preferably <0.2pmol / L, and more preferably <0.1pmol / L.

[0150] Another embodiment of the present application relates to a method according to the aforementioned embodiment, wherein an assay is used to determine the level of PAMP-glycine, wherein the assay sensitivity of the assay is capable of quantifying PAMP-glycine in healthy subjects and is <0.5pmol / L, preferably <0.25pmol / L, and more preferably <0.1pmol / L.

[0151] Another embodiment of the present application relates to a method according to the aforementioned embodiment, wherein an assay is used to determine the level of ADM-Gly, wherein the assay sensitivity of the assay is capable of quantifying ADM-Gly in healthy subjects and is 60 pmol / L, preferably 10 pmol / L, and more preferably 2 pmol / L.

[0152] In a specific embodiment of the invention, the conjugate exhibits at least 10 7 M -1 , preferably 10 8 M -1 The binding affinity is preferably greater than 10 9 M -1 , most preferably above 10 10 M -1 A person skilled in the art will appreciate that it may be considered possible to compensate for the lower affinity by administering a higher dose of the compound, and that such a measure would not result in a departure from the scope of the present invention.

[0153] To determine the affinity of the antibodies for adrenomedullin, the kinetics of adrenomedullin binding to the immobilized antibodies was determined using label-free surface plasmon resonance using a Biacore 2000 system (GE Healthcare Europe GmbH, Freiburg, Germany). Reversible immobilization of the antibodies was performed using anti-mouse Fc antibodies covalently coupled to the surface of a CM5 sensor at high density according to the manufacturer's instructions (Mouse Antibody Capture Kit; GE Healthcare).

[0154] To carry out Lorenz et al., 2011. Antimicrob Agents Chemother. 55(1):165–173 ).

[0155] In a specific embodiment of the present invention, the binder is selected from an antibody or antibody fragment or a non-Ig scaffold that binds to mature ADM-NH2 or proADM and / or fragments thereof.

[0156] In a particular embodiment of the invention, an assay is used to determine the levels of mature ADM-NH2 and / or proADM or fragments thereof having at least 5 amino acids, wherein the assay is a sandwich assay, preferably a fully automated assay.

[0157] In one embodiment of the invention, the testing technology may be a so-called POC test (point-of-care test) which allows testing to be performed near the patient in less than an hour without the need for a fully automated assay system. An example of such a technology is the immunochromatographic testing technology.

[0158] In one embodiment of the invention, the assay is a sandwich immunoassay using any type of detection technology including but not limited to enzyme labels, chemiluminescent labels, electrochemiluminescent labels, preferably a fully automated assay. In one embodiment of the invention, the assay is an enzyme labeled sandwich assay. Examples of automated or fully automated assays include assays that can be used in one of the following systems: Roche Abbott Siemens Brahms Biomerieux Alere Ortho ClinicalDiagnostics

[0159] A variety of different immunoassays are known and can be used in the assays and methods of the present invention, including: radioimmunoassay ("RIA"), homogeneous enzyme multiplication immunoassay ("EMIT"), enzyme-linked immunosorbent assay ("ELISA"), enzyme protein reactivation immunoassay ("ARIS"), test strip immunoassay and immunochromatographic assay.

[0160] In a specific embodiment of the invention, at least one of the two conjugates is labeled so as to be detectable.

[0161] Preferred detection methods include immunoassays in various formats such as radioimmunoassay (RIA), chemiluminescent and fluorescent immunoassays, enzyme-linked immunosorbent assays (ELISA), Luminex-based bead arrays, protein microarray assays, and rapid test formats such as immunochromatographic strip tests.

[0162] In a preferred embodiment, the label is selected from chemiluminescent labels, enzyme labels, fluorescent labels, and radioactive iodine labels.

[0163] The assay may be a homogeneous or heterogeneous assay, a competitive and non-competitive assay. In one embodiment, the assay takes the form of a sandwich assay, which is a non-competitive immunoassay in which the molecule to be detected and / or quantified is bound to a first antibody and a second antibody. The first antibody may be bound to a solid phase such as a bead, a surface, a sheet or a strip of a well or other container, and the second antibody is an antibody labeled with, for example, a dye, a radioisotope or a reactive or catalytically active component. The amount of labeled antibody bound to the analyte is then measured by a suitable method. The general composition and procedures associated with "sandwich assays" have long been established and are known to the skilled artisan ( "immunity The Immunoassay Handbook, edited by David Wild, Elsevier LTD, Oxford; Edition (May 2005); Hultschig et al., 2006. Curr Opin Chem Biol. 10(1):4-10 ).

[0164] In another embodiment, the assay comprises two capture molecules, preferably antibodies, both present as a dispersed system in a liquid reaction mixture, wherein a first labeling component is attached to the first capture molecule, wherein the first labeling component is part of a fluorescence or chemiluminescence-quenching or amplification based labeling system, and a second labeling component of the labeling system is attached to the second capture molecule, such that a measurable signal is generated upon binding of the two capture molecules to the analyte, allowing detection of a sandwich complex formed in a solution containing the sample.

[0165] In another embodiment, the labeling system comprises a rare earth metal cryptate or a rare earth metal chelate in combination with a fluorescent dye or a chemiluminescent dye, in particular a dye of the cyanine type.

[0166] In the context of the present invention, fluorescence-based assays include the use of dyes which may be selected, for example, from FAM (5- or 6-carboxyfluorescein), VIC, NED, fluorescein, fluorescein isothiocyanate (FITC), IRD-700 / 800, cyanine dyes such as CY3, CY5, CY3.5, CY5.5, Cy7, xanthene, 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), TET, 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 5-carboxyrhodamine-6G (R6G5), 6-carboxyrhodamine-6G (RG6), rhodamine, rhodamine green, rhodamine red, rhodamine 110, BODIPY dyes such as BODIPY TMR, Oregon Green, coumarins such as umbelliferone, benzimides such as Hoechst 33258, phenanthridines such as Texas Red, Yakima Yellow, AlexaFluor, PET, ethidium bromide, acridine dyes, carbazole dyes, phenoxazine dyes, porphyrin dyes, polymethine dyes, etc.

[0167] In the context of the present invention, chemiluminescence-based assays involve the use of dyes based on the physical principles described for chemiluminescent materials in the literature ( Kirk-Othmer, Encyclopedia of Chemical Technology Chemical Technology, 4th edition, 1993. John Wiley & Sons, Vol. 15: 518-562, incorporated by reference This article, including citations on pages 551-562 ). Preferred chemiluminescent dyes are acridinium esters.

[0168] When referred to herein, an "assay" or "diagnostic assay" may be any type of assay used in the field of diagnostics. Such an assay may be based on the binding of an analyte to be detected to one or more capture probes with a certain affinity. With respect to the interaction between the capture molecule and the target molecule or molecule of interest, the affinity constant is preferably greater than 10 8 M -1 .

[0169] In the context of the present invention, a "binding molecule" is a molecule that can be used to bind to a target molecule or a molecule of interest, i.e., an analyte (i.e., ADM-NH2 and / or proADM and fragments thereof, in the context of the present invention) from a sample. Therefore, the binding molecule must be fully shaped in terms of both space and surface characteristics such as surface charge, hydrophobicity, hydrophilicity, the presence or absence of Lewis donors and / or receptors to specifically bind to the target molecule or molecule of interest. Therefore, the binding can be mediated, for example, by ionic, van der Waals, π-π, σ-π, hydrophobic or hydrogen bond interactions between the capture molecule and the target molecule or molecule of interest, or a combination of two or more of the above interactions. In the context of the present invention, the binding molecule can be, for example, selected from nucleic acid molecules, carbohydrate molecules, PNA molecules, proteins, antibodies, peptides or glycoproteins. Preferably, the binding molecule is an antibody, including fragments thereof having sufficient affinity to the target molecule or molecule of interest, and including recombinant antibodies or recombinant antibody fragments, as well as chemically and / or biochemically modified derivatives of the antibody or fragments thereof derived from variant chains having a length of at least 12 amino acids.

[0170] The chemiluminescent marker may be an acridinium ester marker, a steroid marker including an isoluminol marker, or the like.

[0171] The enzyme marker can be lactate dehydrogenase (LDH), creatine kinase (CPK), alkaline phosphatase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), acid phosphatase, 6-phosphate glucose dehydrogenase, etc.

[0172] In one embodiment of the invention, at least one of the two conjugates is bound to a solid phase such as magnetic particles, and a polystyrene surface.

[0173] In a specific embodiment of the invention, at least one of the two conjugates is bound to a solid phase.

[0174] In a specific embodiment of the present invention, the threshold value of the ratio of the level of mature ADM-NH2 shown in SEQ ID No.:4 in the body fluid sample of the subject to the level of adrenomedullin precursor or its fragment (which is not the mature ADM-NH2 shown in SEQ ID No.:4) is within a certain range, which is between 0.2 and 0.75, preferably between 0.3 and 0.6, and preferably between 0.4 and 0.5.

[0175] The ADM-NH2 level of the present invention or the level of the corresponding proADM or its fragments is determined using the ADM-NH2 assay (or the corresponding assay for proADM or its fragments) as described in the examples. The threshold values ​​mentioned above may be different in other assays if they are calibrated in a different way than the assay system used in the present invention. Therefore, taking into account the differences in calibration, the critical values ​​mentioned above will be applied accordingly to these differently calibrated assays. One possibility to quantify the calibration differences is to perform a method comparison analysis (correlation) of the assay in question with the corresponding biomarker assay used in the present invention by measuring the corresponding biomarker (e.g. bio-ADM) in the sample using both methods. Another possibility is to use this test to determine the median biomarker level of a representative normal population, compare the results with the median biomarker level described in the literature, and recalculate the calibration based on the differences obtained by this comparison. Samples from normal (healthy) subjects were measured by the calibration used in the present invention: the median plasma bio-ADM (mature ADM-NH2) was 13.7 pg / mL (interquartile range [IQR] 9.6–18.7 pg / mL) ( Weber et al., 2017. JALM, 2(2): 222-233 ).

[0176] Used in Caruhel et al. ( Caruhel et al., 2009. Clin Biochem 42:725-8 ) for the detection of MR-proADM, the median plasma MR-proADM concentration in normal (healthy) subjects was 0.41 (interquartile range 0.23-0.64) nmol / L ( Smith et al., 2009. Clin Chem 55:1593-1595 ).

[0177] The median plasma CT-proADM concentration in normal healthy subjects (n=200) was 77.6 pmol / L (min 46.6 pmol / L, max 136.2 pmol / L) and the 95% percentile was 113.8 pmol / L (EP 2 111 552 B1).

[0178] The plasma concentration of PAMP-amide in normal healthy subjects (n=51) was 0.51±0.19 pmol / L (mean±SD) ( Hashida et al., 2004. Clin Biochem37:14-21 ).

[0179] The plasma concentration of PAMP-glycine in normal healthy subjects (n=51) was 1.15±0.38 pmol / L (mean±SD) ( Hashida et al., 2004. Clin Biochem 37:14-21 ).

[0180] In one embodiment, the threshold may be predetermined as follows:

[0181] comparing the concentration of said marker in a body fluid obtained from said subject to the median value of said marker in body fluid obtained from a predetermined set of samples from a randomly selected population of subjects having comparable baseline conditions to said subject,

[0182] comparing the concentration of said marker in a body fluid obtained from said subject with quantiles of the levels of said marker and / or fragments thereof in body fluid obtained from a set of predetermined samples from a population of subjects having comparable baseline conditions to said subject,

[0183] Calculated on the basis of Cox proportional hazards analysis or by using risk index calculations such as NRI (net reclassification index) or IDI (integrated discrimination index).

[0184] In addition, at least one clinical parameter or biomarker selected from the group consisting of age, race, mental status test (e.g., Mini-Mental State Examination (MMSE)), neuroimaging (CT, MRT, PET, SPECT), family history, ApoE4 genotype, amyloid peptide β1-42 (Aβ 1-42 ), amyloid peptide β1-40 (Aβ 1-40 ), total Tau protein, phosphorylated Tau protein (p-Tau181, p-Tau 199, p-Tau 231).

[0185] In the context of the present invention, the term "dementia" includes Alzheimer's disease, vascular dementia, mixed Alzheimer's disease and vascular dementia, dementia with Lewy bodies, frontotemporal dementia, focal dementias (e.g. progressive aphasia), subcortical dementias (e.g. Parkinson's disease dementia) and secondary causes of dementia syndromes (e.g. intracranial lesions).

[0186] In a more specific embodiment of the invention, said dementia is selected from the group consisting of Alzheimer's disease, vascular dementia and mixed Alzheimer's disease and vascular dementia.

[0187] Most preferably, the dementia is Alzheimer's disease.

[0188] Another subject of the present invention is a pharmaceutical composition comprising the conjugate of the present invention disclosed herein, in particular comprising an anti-ADM antibody or an anti-ADM antibody fragment or an anti-ADM non-Ig backbone for the prevention or treatment of dementia.

[0189] In another embodiment of the present invention, the pharmaceutical composition is a solution, preferably a ready-to-use solution.

[0190] In another embodiment of the present invention, the pharmaceutical composition is a solution, preferably a ready-to-use solution comprising PBS at a pH of 7.4.

[0191] In another embodiment of the present invention, the pharmaceutical composition is in a dry state, which is reconstituted before use.

[0192] In another embodiment of the present invention, the pharmaceutical composition is in a freeze-dried state, which is reconstituted before use.

[0193] In another embodiment of the present invention, the pharmaceutical composition for the prevention and / or treatment of dementia is administered by oral, epidermal, subcutaneous, intradermal, sublingual, intramuscular, intraarterial, intracerebral, intraventricular, intrathecal, intravenous or intraperitoneal administration. In a preferred embodiment of the present invention, the pharmaceutical composition is administered intravenously. In another preferred embodiment of the present invention, the pharmaceutical composition is administered by the central nervous system (CNS), such as intracerebral, intraventricular and intrathecal administration.

[0194] Antibodies according to the present invention are proteins that specifically bind antigens, and include one or more polypeptides that are substantially encoded by immunoglobulin genes. Recognized immunoglobulin genes include κ, λ, α (IgA), γ (IgG1, IgG2, IgG3, IgG4), δ (IgD), ε (IgE) and μ (IgM) constant region genes and countless immunoglobulin variable region genes. Full-length immunoglobulin light chain length is generally about 25Kd or 214 amino acids. Full-length immunoglobulin heavy chain length is generally about 50Kd or 446 amino acids. Light chain is encoded by variable region genes at NH2-end (length is about 110 amino acids), and is encoded by κ or λ constant region genes at COOH-end. Similarly, heavy chain is encoded by variable region genes (length is about 116 amino acids) and one of other constant region genes.

[0195] The basic structural unit of an antibody is usually a tetramer, which is composed of two identical pairs of immunoglobulin chains, each pair having one light chain and one heavy chain. In each pair, the light and heavy chain variable regions bind to the antigen, and the constant regions mediate effector functions. Immunoglobulins also exist in a variety of other forms, such as Fv, Fab and (Fab')2, as well as bifunctional hybrid antibodies and single chains (e.g. Lanzavecchia et al., 1987. Eur. J. Immunol. 17:105; Huston et al., 1988, Proc. Natl. Acad. Sci. USA, 85:5879-5883; Bird et al., 1988, Science242:423-426; Hood et al., Immunology, Benjamin, NY, 2nd ed., 1984; Hunkapiller and Hood 1986. Nature 323:15-16 The variable region of an immunoglobulin light or heavy chain consists of a framework region interrupted by three hypervariable regions, also called complementarity determining regions (CDRs) (see Sequences of Proteins of Immunological Interest, E. Kabat et al., USDepartment of Health and Human Services, 1983 ). As mentioned above, CDR is mainly responsible for binding to antigen epitope. Immune complex is an antibody such as monoclonal antibody, chimeric antibody, humanized antibody or human antibody or functional antibody fragment that specifically binds to antigen.

[0196] Chimeric antibodies are antibodies whose light and heavy chain genes are constructed from immunoglobulin variable and constant region genes belonging to different species, usually by genetic engineering. For example, the variable segments of the genes from a mouse monoclonal antibody can be linked to human constant segments such as κ and γ1 or γ3. Thus, in one example, a therapeutic chimeric antibody is a hybrid protein consisting of a variable or antigen-binding domain from a mouse antibody and a constant or effector domain from a human antibody, although other mammalian species may be used or the variable regions may be produced by molecular techniques. Methods for making chimeric antibodies are well known in the art (see, for example, U.S. Patent No. 5,807,715 ). A "humanized" immunoglobulin is an immunoglobulin that includes a human framework region and one or more CDRs from a non-human (e.g., mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin that provides the CDRs is called a "donor," and the human immunoglobulin that provides the framework is called an "acceptor." In one embodiment, all CDRs in the humanized immunoglobulin come from a donor immunoglobulin. Constant regions do not necessarily have to be present, but if they are present, they must be substantially consistent with human immunoglobulin constant regions, i.e., at least about 85-90%, such as about 95% or more consistent. Therefore, all parts of the humanized immunoglobulin, except for possible CDRs, are substantially consistent with corresponding parts of natural human immunoglobulin sequences. A "humanized antibody" is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of replacements of amino acids taken from the donor framework. Humanized or other monoclonal antibodies may have additional conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions. Exemplary conservative substitutions are, for example, gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr. Humanized immunoglobulins can be constructed using genetic engineering (see, for example, U.S. Patent No. 5,585,089 ). Human antibodies are antibodies in which both the light chain and heavy chain genes are of human origin. Human antibodies can be produced using methods known in the art. Human antibodies can be produced by immortalizing human B cells that secrete the antibody of interest. Immortality can be achieved, for example, by EBV infection or by fusing human B cells with myeloma or hybridoma cells to produce tri-source tumor cells. Human antibodies can also be produced by phage display methods (see, e.g. Dower et al., PCT Publication No. WO91 / 17271; McCafferty et al., PCT Publication No. WO92 / 001047; and Winter, PCT Publication No. WO92 / 20791 ) or selected from a human combinatorial monoclonal antibody library (see the Morphosys website). Human antibodies can also be prepared using transgenic animals carrying human immunoglobulin genes (see, for example, Lonberg et al., PCT Publication No. WO 93 / 12227; and Kucherlapati, PCT Publication No. WO 91 / 10741).

[0197] Therefore, the antibody can have a form known in the art. Examples are human antibodies, monoclonal antibodies, humanized antibodies, chimeric antibodies, CDR-grafted antibodies. In a preferred embodiment, the antibody according to the present invention is a recombinantly produced antibody such as IgG, typically a full-length immunoglobulin, or an antibody fragment containing at least the F-variable domain of a heavy chain and / or a light chain, such as a chemically coupled antibody (antigen binding fragment), including but not limited to Fab-fragments including Fab miniantibodies, single-chain Fab antibodies, monovalent Fab antibodies with epitope tags such as Fab-V5Sx2, bivalent Fab (miniantibodies) dimerized with a CH3 domain, bivalent Fab or multivalent Fab such as Fab-dHLX-FSx2, F(ab')2-fragments, scFv-fragments, multimerized multivalent or / and multispecific scFv-fragments, bivalent and / or bispecific diabodies, (bispecific T-cell engagers), triabodies, multivalent antibodies such as those from classes other than G, single domain antibodies such as nanobodies derived from camelid or fish immunoglobulins, and a large number of other antibodies or fragments.

[0198] Besides antibodies, other biopolymer backbones for complexing target molecules are known in the art and have been used to generate highly target-specific biopolymers. Examples are aptamers, spiegelmers, anticalins and conotoxins.

[0199] In a preferred embodiment, the antibody form is selected from Fv fragments, scFv fragments, Fab fragments, scFab fragments, (Fab)2 fragments and scFv-Fc fusion proteins. In another preferred embodiment, the antibody form is selected from scFab fragments, Fab fragments, scFv fragments and bioavailability optimized conjugates thereof such as PEGylated fragments. One of the most preferred forms is the scFab form.

[0200] The non-Ig scaffolds may be protein scaffolds and may be used as antibody mimics because they are able to bind to ligands or antigens. The non-Ig scaffolds may be selected from tetranectin-based non-Ig scaffolds (e.g., US2010 / 0028995 ), fibronectin backbones (e.g., EP 1 266 025 ), lipocalin-based scaffolds (e.g., WO 2011 / 154420 described in ), the ubiquitin backbone (e.g. WO 2011 / 073214 described in ), transfer backbones (e.g. US 2004 / 0023334), protein A backbone (e.g., EP 2 231 860 ), ankyrin repeat-based backbones (e.g., WO 2010 / 060748 ), a microprotein backbone (preferably a microprotein forming a cystine knot) (e.g. EP 2 314 308 ), scaffolds based on the Fyn SH3 domain (e.g., WO 2011 / 023685 ), EGFR-A domain-based scaffolds (e.g., WO 2005 / 040229 ) and Kunitz domain-based scaffolds (e.g., EP 1941867 described in ).

[0201] Furthermore, in one embodiment of the present invention, the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is monospecific.

[0202] A monospecific anti-adrenomedullin (ADM) antibody or monospecific anti-adrenomedullin antibody fragment or monospecific anti-ADM non-Ig scaffold means that the antibody or antibody fragment or non-Ig scaffold binds to a specific region covering at least 5 amino acids within the target ADM. A monospecific anti-adrenomedullin (ADM) antibody or monospecific anti-adrenomedullin antibody fragment or monospecific anti-ADM non-Ig scaffold is an anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold that all have affinity for the same antigen.

[0203] In another specific and preferred embodiment, the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig backbone that binds to ADM is a monospecific antibody, antibody fragment or non-Ig backbone, respectively, wherein monospecific means that the antibody or antibody fragment or non-Ig backbone binds to a specific region covering at least 4 amino acids within the target ADM. The monospecific antibody or fragment or non-Ig backbone according to the present invention is an antibody or fragment or non-Ig backbone that all has affinity for the same antigen. Monoclonal antibodies are monospecific, but monospecific antibodies can also be produced by other means other than production from common germ cells.

[0204] The anti-ADM antibody or antibody fragment that binds to ADM or the non-Ig scaffold that binds to ADM may be a non-neutralizing anti-ADM antibody or antibody fragment that binds to ADM or the non-Ig scaffold that binds to ADM.

[0205] In a specific embodiment, the anti-ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig backbone is a non-neutralizing antibody, fragment or non-Ig backbone. Neutralizing anti-ADM antibodies, anti-ADM antibody fragments or anti-ADM non-Ig backbones block the biological activity of ADM by almost 100%, at least more than 90%, preferably at least more than 95%.

[0206] In contrast, the non-neutralizing anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig backbone blocks the biological activity of ADM by less than 100%, preferably less than 95%, preferably less than 90%, more preferably less than 80%, and even more preferably less than 50%. This means that the biological activity of ADM is reduced by less than 100%, less than 95% or less and not more, less than 90% or less and not more, less than 80% or less and not more, less than 50% or less and not more. This means that the residual biological activity of ADM bound to the non-neutralizing anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig backbone will exceed 0%, preferably exceed 5%, preferably exceed 10%, more preferably exceed 20%, and more preferably exceed 50%.

[0207] In this context, for simplicity, antibody fragments or non-Ig scaffolds of "non-neutralizing" anti-ADM antibodies are collectively referred to herein as "non-neutralizing" anti-ADM antibodies, such as antibodies or antibody fragments or non-Ig scaffolds that block the biological activity of ADM by less than 80%, and are defined as:

[0208] One or more molecules that bind to ADM, which, upon addition to a culture of a eukaryotic cell line expressing a functional human recombinant ADM receptor composed of CRLR (calcitonin receptor-like receptor) and RAMP3 (receptor activity modifying protein 3), reduce the amount of cAMP produced by the cell line through the action of a human synthetic ADM peptide added in parallel, wherein the added human synthetic ADM is added in an amount that causes half-maximal stimulation of cAMP synthesis in the absence of the non-neutralizing antibody to be analyzed, wherein the reduction of cAMP by the molecule that binds to ADM occurs to an extent of no more than 80%, even when the non-neutralizing molecule that binds to ADM to be analyzed is added in an amount that is 10 times higher than the amount required to obtain the maximal reduction in cAMP synthesis that can be obtained using the non-neutralizing antibody to be analyzed.

[0209] The same definition applies to other ranges: 95%, 90%, 50%, etc.

[0210] In a preferred embodiment of the present invention, the anti-ADM antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold binds to a region or epitope of ADM located in the N-terminal portion (aa 1-21) of adrenomedullin.

[0211] In another preferred embodiment, the anti-ADM antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold recognizes and binds to a region or epitope within amino acids 1-14 of adrenomedullin (SEQ ID No.: 27); this means the N-terminal portion of adrenomedullin (aa 1-14). In another preferred embodiment, the anti-ADM antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold recognizes and binds to a region or epitope within amino acids 1-10 of adrenomedullin (SEQ ID No.: 28); this means the N-terminal portion of adrenomedullin (aa 1-10).

[0212] ADM aa 1-14

[0213] YRQSMNNFQGLRSF (SEQ ID No.: 27)

[0214] ADM aa 1-10

[0215] YRQSMNNFQG (SEQ ID No.: 28)

[0216] In another preferred embodiment, the anti-ADM antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig backbone recognizes and binds to a region or epitope within amino acids 1-6 of adrenomedullin (SEQ ID No.: 29); this means the N-terminal portion of adrenomedullin (aa 1-6). As described above, the region or epitope preferably comprises a length of at least 4 or at least 5 amino acids.

[0217] ADM aa 1-6

[0218] YRQSMN (SEQ ID No.: 29)

[0219] In another preferred embodiment, the anti-ADM antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold recognizes and binds to the N-terminal end (aa1) of adrenomedullin. The N-terminal end means the 1st amino acid, which is "Y" of SEQ ID No.: 4, 5, 21, 27, 28 or 29; it is mandatory for antibody binding. The antibody or fragment or scaffold binds neither to adrenomedullin with N-terminal extension or N-terminal modification nor to adrenomedullin with N-terminal degradation. This means that in another preferred embodiment, if the N-terminal end of ADM is not present, the anti-ADM antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold only binds to the region within the mature ADM sequence. In the embodiment, if the sequence is contained in, for example, pro-ADM, the anti-ADM antibody or anti-adrenomedullin antibody fragment or non-Ig scaffold will not bind to the region within the mature ADM sequence.

[0220] For the sake of clarity, numbers in brackets for specific regions of ADM such as "N-terminal portion (aa 1-21)" are understood by those skilled in the art to mean that the N-terminal portion of ADM consists of amino acids 1-21 of the mature ADM sequence.

[0221] In one embodiment of the present invention, the antibody according to the present invention can be produced as follows:

[0222] Balb / c mice were immunized with ADM-100 μg peptide-BSA-conjugate (emulsified in 100 μl Freund's complete adjuvant) on days 0 and 14 and with 50 μg (in 100 μl Freund's incomplete adjuvant) on days 21 and 28. Three days before the fusion experiments, the animals received 50 μg of the conjugate dissolved in 100 μl saline, given as one intraperitoneal and one intravenous injection.

[0223] Splenocytes from the immunized mice were fused with cells of myeloma cell line SP2 / 0 using 1 ml of 50% polyethylene glycol at 37° C. for 30 s. After washing, cells were seeded in 96-well cell culture plates. Hybrid clones were selected by growing in HAT medium [RPMI 1640 medium supplemented with 20% fetal bovine serum and HAT supplement]. After two weeks, the HAT medium was replaced with HT medium for three passages and then returned to normal cell culture medium.

[0224] Three weeks after fusion, cell culture supernatants were screened for antigen-specific IgG antibodies. Microcultures that tested positive were transferred to 24-well plates for proliferation. After retesting, selected cultures were cloned and recloned using limiting dilution techniques and isotyped (see also Lane 1985. J. Immunol. Meth. 81: 223-228; Ziegler, B. et al., 1996 Horm.Metab.Res.28:11-15 ).

[0225] Antibodies can be produced using phage display technology according to the following procedure:

[0226] The human naive antibody gene library HAL7 / 8 was used to isolate recombinant single chain F-variable domains (scFv) against the adrenomedullin peptide. The antibody gene library was screened using a panning strategy that included the use of peptides containing a biotin tag linked to the adrenomedullin peptide sequence via two different spacers. A mixture of panning rounds using nonspecifically bound antigen and streptavidin-bound antigen was used to minimize the background of nonspecific binders. Phage eluted from the third round of panning was used to generate E. coli strains expressing monoclonal scFv. Supernatants from the cultures of these clones were used directly for antigen ELISA testing (see Hust et al., 2011. Journal of Biotechnology 152: 159–170;Schütte et al., 2009.PLoS One4,e6625 ).

[0227] Humanization of murine antibodies can be performed according to the following procedure:

[0228] In order to humanize an antibody of murine origin, the structural interaction of the framework regions with complementary determining regions (CDRs) of the antibody sequence with the antigen is analyzed. Suitable FRs of human origin are selected on the basis of structural modeling, and the murine CDR sequences are transplanted into the human FRs. Mutations can be introduced in the amino acid sequence of CDRs or FRs to regain structural interactions that were abolished by species switching of FR sequences. This restoration of structural interactions can be achieved by random methods using phage display libraries or by directed methods guided by molecular modeling ( Almagro et al., 2008. Front Biosci.2008;13:1619-33 ).

[0229] Another embodiment of the present application relates to a method according to the aforementioned embodiment, wherein the anti-ADM antibody that binds to the N-terminal portion aa 1-21 of adrenomedullin for treating a subject is a human CDR-grafted antibody or an antibody fragment thereof that binds to ADM, wherein the human CDR-grafted antibody or an antibody fragment thereof comprises an antibody heavy chain (H chain) comprising:

[0230] SEQ ID NO.7:

[0231] GYTFSRYW

[0232] SEQ ID NO.:8:

[0233] ILPGSGST

[0234] and / or

[0235] SEQ ID NO.:9:

[0236] TEGYEYDGFDY

[0237] and / or further comprises an antibody light chain (L chain), comprising:

[0238] SEQ ID NO.: 10:

[0239] QSIVYSNGNTY

[0240] SEQ ID NO.: 11: (not mentioned in the sequence listing due to its length of 3 amino acids)

[0241] RVS

[0242] and / or

[0243] SEQ ID NO.: 12:

[0244] FQGSHIPYT.

[0245] In another specific embodiment of the present application, the anti-ADM antibody for treating a subject is a human monoclonal antibody or an antibody fragment thereof that binds to ADM, wherein the heavy chain comprises at least one CDR selected from the following:

[0246] SEQ ID NO.:7:

[0247] GYTFSRYW

[0248] SEQ ID NO.:8:

[0249] ILPGSGST

[0250] SEQ ID NO.:9:

[0251] TEGYEYDGFDY

[0252] And wherein the light chain comprises at least one CDR selected from:

[0253] SEQ ID NO.: 10:

[0254] QSIVYSNGNTY

[0255] SEQ ID NO.: 11: (not mentioned in the sequence listing due to its length of 3 amino acids)

[0256] RVS

[0257] SEQ ID NO.: 12:

[0258] FQGSHIPYT.

[0259] In another embodiment of the present application, the anti-ADM antibody for treating a subject is a human monoclonal antibody or an antibody fragment thereof that binds to ADM, wherein the heavy chain comprises the sequence:

[0260] SEQ ID NO.:7:

[0261] GYTFSRYW

[0262] SEQ ID NO.:8:

[0263] ILPGSGST

[0264] SEQ ID NO.:9:

[0265] TEGYEYDGFDY

[0266] and wherein the light chain comprises the sequence:

[0267] SEQ ID NO.: 10:

[0268]

[0269] SEQ ID NO.: 11: (not mentioned in the sequence listing due to its length of 3 amino acids)

[0270]

[0271] SEQ ID NO.: 12:

[0272]

[0273] Another embodiment of the present application relates to the method of the preceding embodiment, wherein the antibody or fragment used for treatment is a human monoclonal antibody or an antibody fragment thereof that binds to ADM, wherein the heavy chain comprises the sequence:

[0274] CDR1: SEQ ID No.: 7:

[0275]

[0276] CDR2: SEQ ID No.: 8:

[0277]

[0278] CDR3: SEQ ID No.: 9:

[0279]

[0280] and wherein the light chain comprises the sequence:

[0281] CDR1: SEQ ID No.: 10:

[0282]

[0283] CDR2: SEQ ID No.: 11: (not mentioned in the sequence listing due to its length of 3 amino acids)

[0284]

[0285] CDR3: SEQ ID No.: 12:

[0286]

[0287] Another embodiment of the present application relates to the method of the aforementioned embodiment, wherein the antibody or fragment for treatment comprises the following sequence as the VH region:

[0288] SEQ ID NO.: 13 (AM-VH-C):

[0289]

[0290] SEQ ID NO.: 14 (AM-VH1):

[0291]

[0292] SEQ ID NO.: 15 (AM-VH2-E40):

[0293]

[0294]

[0295] SEQ ID NO.:16(AM-VH3-T26-E55):

[0296]

[0297] SEQ ID NO.:17(AM-VH4-T26-E40-E55):

[0298]

[0299] And comprises the following sequence as VL region:

[0300] SEQ ID NO.: 18 (AM-VL-C):

[0301]

[0302] SEQ ID NO.: 19 (AM-VL1):

[0303]

[0304] SEQ ID NO.: 20 (AM-VL2-E40):

[0305]

[0306] The following embodiments are subject of the present invention:

[0307] 1) A method for:

[0308] a) diagnosis of dementia, or

[0309] b) determine the risk of developing dementia in a subject who does not have dementia, or

[0310] c) monitoring treatment of a subject with dementia or monitoring or directing interventions for a subject with dementia, or

[0311] d) monitoring treatment of subjects at risk of developing dementia or monitoring or directing preventive interventions for subjects at risk of developing dementia,

[0312] wherein the level of mature ADM-NH2 shown in SEQ ID No.: 4 in a body fluid sample of the subject is determined, and wherein the level of mature ADM-NH2 is compared with a threshold level,

[0313] And among them

[0314] a) if the level of mature ADM-NH2 shown in SEQ ID No.: 4 is below the threshold level, the subject is diagnosed as having dementia, or wherein

[0315] b) if the level of mature ADM-NH2 shown in SEQ ID No.: 4 is below the threshold level, the subject has an increased risk of developing dementia, or wherein

[0316] c) if the level of mature ADM-NH2 shown in SEQ ID No.:4 increases during the treatment or intervention, the condition of the subject suffering from dementia or at risk of dementia is improving under the treatment or intervention, and / or if the level of mature ADM-NH2 shown in SEQ ID No.:4 increases to above the threshold value, the intervention can continue.

[0317] 2) A method for:

[0318] a) diagnosis of dementia, or

[0319] b) determine the risk of developing dementia in a subject who does not have dementia, or

[0320] c) monitoring treatment of a subject with dementia or monitoring or directing interventions for a subject with dementia, or

[0321] d) monitoring preventive treatment of subjects at risk of developing dementia or monitoring or directing preventive interventions in subjects at risk of developing dementia,

[0322] wherein a marker ratio is determined, the marker ratio may be a ratio of the determined level of mature ADM-NH2 shown in SEQ ID No.: 4 in the subject's body fluid sample to the determined level of adrenomedullin precursor or a fragment thereof (which is not mature ADM-NH2 shown in SEQ ID No.: 4) in the subject's body fluid sample, and wherein the marker ratio is compared with a ratio threshold value,

[0323] And among them

[0324] a) if the marker ratio of mature ADM-NH2 / adrenomedullin precursor or a fragment thereof is below the ratio threshold, the subject is diagnosed as having dementia, or wherein

[0325] b) if the ratio of the marker of mature ADM-NH2 / adrenomedullin precursor or a fragment thereof is below the ratio threshold, the subject has an increased risk of suffering from dementia, or wherein

[0326] c) if the ratio of said markers increases during the course of the treatment or intervention, the condition of the subject suffering from dementia or at risk of developing dementia is improving under the treatment or intervention,

[0327] Alternatively, instead of the above-mentioned marker ratio, the level of mature ADM-NH2 shown in SEQ ID No.:4 in the body fluid sample of the subject is determined and the level of adrenomedullin precursor or a fragment thereof (which is not the mature ADM-NH2 shown in SEQ ID No.:4) in the body fluid sample of the subject is determined, and the two determined levels are combined in a mathematical algorithm, wherein the result of the mathematical algorithm is used to diagnose dementia, or to determine the risk of dementia in a subject without dementia, or to monitor the treatment of a subject with dementia, or to monitor or guide intervention for a subject with dementia, or to monitor preventive treatment for a subject at risk of dementia, or to monitor or guide preventive intervention for a subject at risk of dementia.

[0328] 3. The method according to item 2, wherein the fragment of the adrenomedullin precursor is selected from PAMP (SEQ ID No.: 2), MR-proADM (SEQ ID No.: 3), ADM-Gly (SEQ ID No.: 5) and CT-proADM (SEQ ID No.: 6).

[0329] 4. The method according to item 1, wherein the threshold level of mature ADM-NH2 shown in SEQ ID No.: 4 is equal to or lower than 15 pg / mL, preferably equal to or lower than 10 pg / mL, preferably equal to or lower than 5 pg / mL.

[0330] 5. The method according to item 2 or 3, wherein the ratio threshold is in the range of 0.2 to 0.75, preferably 0.3 to 0.6, preferably 0.4 to 0.5.

[0331] 6. The method according to any one of the preceding items, wherein the sample is selected from the group consisting of blood, serum, plasma, urine, cerebrospinal fluid (CSF) and saliva.

[0332] 7. The method according to items 1 to 6, wherein the body fluid sample is obtained from a subject who has never been diagnosed with dementia or MCI at the time of sampling.

[0333] 8. The method according to any of the preceding items, wherein at least one additional clinical parameter selected from the group consisting of age, race, mental status test (e.g. Mini-Mental State Examination (MMSE)), neuroimaging (CT, MRT, PET, SPECT), family history, ApoE4 genotype, amyloid peptide β1-42 (Aβ 1-42 ), amyloid peptide β1-40 (Aβ 1-40 ), total Tau protein, phosphorylated Tau protein (p-Tau 181, p-Tau 199, p-Tau 231).

[0334] 9. The method according to any of the preceding items, wherein the level of the marker is determined by immunoassay.

[0335] 10. The method according to any of the preceding items, wherein the method is used for patient stratification to select patients for treatment with an anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold for preventing or treating dementia in a subject, wherein the anti-ADM antibody or anti-ADM fragment or anti-ADM non-Ig scaffold binds to the N-terminal portion (aa 1-21) of adrenomedullin:

[0336] YRQSMNNFQGLRSFGCRFGTC (SEQ ID No.: 21).

[0337] 11. A use of an anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or an anti-ADM non-Ig scaffold for preventing and treating dementia in a subject, wherein the anti-ADM antibody or anti-ADM fragment or anti-ADM non-Ig scaffold binds to the N-terminal portion (aa 1-21) of adrenomedullin:

[0338] YRQSMNNFQGLRSFGCRFGTC (SEQ ID No.: 21).

[0339] 12. Use of the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig skeleton according to item 11 in preventing and treating dementia in a subject, wherein the level of mature ADM-NH2 shown in SEQ ID No.:4 determined in the subject's body fluid sample is lower than a threshold level, and / or the subject has a marker ratio, which is the ratio of the determined level of mature ADM-NH2 shown in SEQ ID No.:4 in the subject's body fluid sample to the determined level of adrenomedullin precursor or its fragment in the subject's body fluid sample, and wherein the marker level ratio is lower than the ratio threshold.

[0340] 13. Use of the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig skeleton according to item 12 for preventing and treating dementia in a subject, wherein the fragment of the adrenomedullin precursor is selected from PAMP (SEQ ID No.: 2), MR-proADM (SEQ ID No.: 3), ADM-Gly (SEQ ID No.: 5) and CT-proADM (SEQ ID No.: 6).

[0341] 14. Use of the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig skeleton according to items 11 to 13 for preventing and treating dementia in a subject, wherein the subject is selected by the method according to item 10.

[0342] 15. Use of an anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or an anti-ADM non-Ig skeleton according to any one of items 12 to 14 in the prevention and treatment of dementia in a subject, wherein the threshold level of mature ADM-NH2 shown in SEQ ID No.: 4 is equal to or lower than 15 pg / mL, preferably equal to or lower than 10 pg / mL, preferably equal to or lower than 5 pg / mL.

[0343] 16. Use of the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig skeleton according to items 12 to 14 for preventing and treating dementia in a subject, wherein the marker level ratio is in the range of 0.2 to 0.75, preferably 0.3 to 0.6, preferably 0.4 to 0.5.

[0344] 17. Use of the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig skeleton according to items 11 to 16 for preventing and treating dementia in a subject, wherein the subject is selected according to the method described in item 10, and wherein the body fluid sample is selected from blood, serum, plasma, urine, cerebrospinal fluid (CSF) and saliva.

[0345] 18. Use of an anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or an anti-ADM non-Ig backbone according to items 11 to 16 for the prevention and treatment of dementia in a subject, wherein at least one additional clinical parameter selected from the group consisting of age, race, mental status test (e.g. Mini-Mental State Examination (MMSE)), neuroimaging (CT, MRT, PET, SPECT), family history, ApoE4 genotype, amyloid peptide β1-42 (Aβ 1-42 ), amyloid peptide β1-40 (Aβ 1-40 ), total Tau protein, phosphorylated Tau protein (p-Tau 181, p-Tau 199, p-Tau231).

[0346] 19. Use of the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig backbone according to items 12 to 18 for preventing and treating dementia in a subject, wherein the level of the marker is determined by immunoassay.

[0347] 20. Use of an anti-adrenomedullin (ADM) antibody or an anti-ADM antibody fragment or an anti-ADM non-Ig scaffold binding to adrenomedullin according to items 12 to 19 for preventing and treating dementia in a subject, wherein the antibody or fragment or scaffold exhibits at least 10% affinity for ADM. -7 The binding affinity of M.

[0348] 21. Use of the anti-ADM antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold according to items 12 to 20 for preventing and treating dementia in a subject, wherein the antibody or fragment or scaffold recognizes and binds to the N-terminal end (aa 1) of adrenomedullin.

[0349] 22. Use of an anti-ADM antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig skeleton according to items 12 to 21 for preventing and treating dementia in a subject, wherein the antibody or fragment or skeleton blocks the biological activity of ADM by no more than 80%, preferably no more than 50%.

[0350] Example

[0351] Example 1

[0352] Generation of antibodies and determination of their affinity constants

[0353] Several human and murine antibodies were generated and their affinity constants determined (see Table 1).

[0354] Peptides / conjugates for immunization:

[0355] Referring to Table 1, peptides for immunization were synthesized (JPT Technologies, Berlin, Germany) with an additional N-terminal cysteine ​​(if cysteine ​​is not present in the selected ADM sequence) residue for coupling the peptide to bovine serum albumin (BSA). The peptide was covalently linked to BSA using Sulfolink-coupling gel (Perbio Science, Bonn, Germany). The coupling procedure was performed according to the manual of Perbio.

[0356] Murine antibodies were generated as follows:

[0357] Balb / c mice were immunized with 100 μg of peptide-BSA-conjugate (emulsified in 100 μl Freund's complete adjuvant) on days 0 and 14 and 50 μg (in 100 μl Freund's incomplete adjuvant) on days 21 and 28. Three days before the fusion experiments, the animals received 50 μg of the conjugate dissolved in 100 μl saline, given as one intraperitoneal and one intravenous injection.

[0358] Splenocytes from the immunized mice were fused with cells of myeloma cell line SP2 / 0 using 1 ml of 50% polyethylene glycol at 37° C. for 30 s. After washing, cells were seeded in 96-well cell culture plates. Hybrid clones were selected by growing in HAT medium [RPMI 1640 medium supplemented with 20% fetal bovine serum and HAT supplement]. After two weeks, the HAT medium was replaced with HT medium for three passages and then returned to normal cell culture medium.

[0359] Three weeks after fusion, cell culture supernatants were first screened for antigen-specific IgG antibodies. Microcultures that tested positive were transferred to 24-well plates for proliferation. After retesting, selected cultures were cloned and recloned using limiting dilution techniques and isotyped (see also Lane, RD1985.J.Immunol.Meth.81:223-228; Ziegler et al., 1996.Horm.Metab.Res.28:11-15 ).

[0360] Mouse monoclonal antibody production:

[0361] Antibodies were produced by standard antibody production methods ( Marx et al., 1997, Monoclonal Antibody Production, ATLA 25, 121 ) and purified by protein A. Based on SDS gel electrophoresis analysis, the antibody purity was >95%.

[0362] Human Antibodies:

[0363] Human antibodies were produced using phage display technology according to the following procedure:

[0364] The human naive antibody gene library HAL7 / 8 is used to separate the recombinant single-chain F-variable domain (scFv) for the ADM peptide. The antibody gene library is screened with a panning strategy, which includes the use of a peptide containing a biotin tag connected to the ADM peptide sequence by two different spacers. A mixture of a panning round of antigens bound to nonspecific binding and streptavidin is used to minimize the background of nonspecific binding. The phages eluted from the third round of panning are used to produce an E. coli strain expressing a monoclonal scFv. The supernatant from the culture of these clones is directly used for antigen ELISA testing (see Hust et al., 2011. Journal of Biotechnology 152:159–170; Schütte et al., 2009.PLoS One4,e6625 ).

[0365] Positive clones were selected based on positive ELISA signals against antigen and negative signals against streptavidin-coated microtiter plates. For further characterization, the scFv open reading frame was cloned into the expression plasmid pOPE107 ( Hust et al., 2011. Journal of Biotechnology152,159–170 ), captured from culture supernatant by immobilized metal ion affinity chromatography, and purified by size exclusion chromatography.

[0366] Affinity constant:

[0367] To determine the affinity of the antibody for ADM, the kinetics of ADM binding to the immobilized antibody was determined using label-free surface plasmon resonance using a Biacore 2000 system (GE Healthcare Europe GmbH, Freiburg, Germany). Reversible immobilization of the antibody was performed using an anti-mouse Fc antibody covalently coupled to the surface of a CM5 sensor at high density according to the manufacturer's instructions (Mouse Antibody Capture Kit; GE Healthcare). Lorenz et al., 2011. Antimicrob Agents Chemother. 55(1): 165–173 ).

[0368] Monoclonal antibodies were generated against the ADM region of the following human and murine ADM, respectively. The following table represents a selected set of resulting antibodies used in further experiments. The selection was based on the target region:

[0369] Table 1:

[0370]

[0371] Generation of antibody fragments by enzymatic digestion:

[0372] The production of Fab and F(ab)2 fragments was performed by enzymatic digestion of the murine full-length antibody NT-M. Antibody NT-M was digested with a) a pepsin-based F(ab)2 preparation kit (Pierce 44988) and b) a papain-based Fab preparation kit (Pierce 44985). The fragmentation procedure was performed according to the instructions provided by the supplier. In the case of F(ab)2 fragmentation, digestion was performed at 37°C for 8 h. Fab fragmentation digestion was performed for 16 h accordingly.

[0373] Procedure for Fab production and purification:

[0374] The immobilized papain was balanced by washing the resin with 0.5ml digestion buffer and centrifuging the column at 5000x g for 1 minute. The buffer was then discarded. The desalting column was prepared by removing the storage solution and washing it with digestion buffer, and then centrifuging it at 1000x g for 2 minutes each time. The IgG sample prepared by 0.5ml was added to the centrifuge column tube containing the balanced immobilized papain. The incubation time of the digestion reaction was 16h at 37°C on a tabletop shaker. The column was centrifuged at 5000×g for 1 minute to separate the digest from the immobilized papain. The resin was then washed with 0.5ml PBS and centrifuged at 5000×g for 1 minute. The cleaning fraction was added to the digested antibody, and the total sample volume was 1.0ml. The NAb protein A column was balanced at room temperature with PBS and IgG elution buffer. The column was centrifuged for 1 minute to remove the storage solution (containing 0.02% sodium azide) and balanced by adding 2ml PBS, centrifuged again for 1 minute and the flow-through was discarded. The sample was applied to the column and resuspended by inversion. Incubation was performed at room temperature for 10 minutes with inversion mixing. The column was centrifuged for 1 minute and the flow-through with the Fab fragments was retained. (References: Coulter and Harris1983.J.Immunol.Meth.59,199-203.; Lindner et al., 2010. Cancer Res. 70, 277-87; Kaufmann et al., 2010. PNAS. 107, 18950-5.; Chen et al., 2010.PNAS.107,14727-32; Uysal et al., 2009 J.Exp.Med.206,449-62; Thomas et al., 2009.J.Exp.Med.206,1913-27; Kong et al., 2009 J.Cell Biol.185,1275-840 ).

[0375] Methods for F(ab')2 fragment production and purification:

[0376] The immobilized pepsin was balanced by washing the resin with 0.5ml digestion buffer and centrifuging the column at 5000x g for 1 minute. The buffer was then discarded. The desalting column was prepared by removing the storage solution and washing it with digestion buffer, and then centrifuging it at 1000x g for 2 minutes each time. 0.5ml of the prepared IgG sample was added to the centrifuge column tube containing the balanced immobilized pepsin. The incubation time of the digestion reaction was 16h at 37°C on a tabletop shaker. The column was centrifuged at 5000×g for 1 minute to separate the digest from the immobilized pepsin. The resin was then washed with 0.5mL PBS and centrifuged at 5000×g for 1 minute. The cleaning fraction was added to the digested antibody, and the total sample volume was 1.0ml. The NAb protein A column was balanced at room temperature with PBS and IgG elution buffer. The column was centrifuged for 1 minute to remove the storage solution (containing 0.02% sodium azide) and balanced by adding 2ml PBS, centrifuged again for 1 minute and the flow-through was discarded. The sample was applied to the column and resuspended by inversion. The incubation was carried out at room temperature for 10 minutes with inversion mixing. The column was centrifuged for 1 minute and the flow-through with the Fab fragments was retained. (References: Mariani et al., 1991. Mol. Immunol. 28:69-77; Beale1987. Exp Comp Immunol 11:287-96; Ellerson et al., 1972. FEBS Letters 24(3):318-22; Kerbel and Elliot1983. Meth Enzymol93:113-147; Kulkarni et al., 1985. Cancer Immunol Immunotherapy19:211-4; Lamoyi 1986. Meth Enzymol121:652-663; Parham et al., 1982.J Immunol Meth53:133-73; Raychaudhuri et al., 1985. Mol Immunol22(9):1009-19; Rousseaux et al., 1980. Mol Immunol 17:469-82; Rousseaux et al., 1983. J Immunol Meth 64:141-6; Wilson et al., 1991.J Immunol Meth138:111-9 ).

[0377] Humanization of NT-H antibody fragment:

[0378] Antibody fragments were humanized by CDR grafting ( Jones et al., 1986. Nature 321, 522–525 ).

[0379] To obtain the humanized sequence the following steps were performed:

[0380] -Total RNA extraction: Total RNA was extracted from NT-H hybridoma using Qiagen kit.

[0381] -First round RT-PCR: Use One-step RT-PCR kit (Cat. No. 210210). RT-PCR was performed using a primer set specific for heavy chain and light chain. For each RNA sample, 12 separate heavy chain and 11 light chain RT-PCR reactions were set up using a degenerate forward primer mixture covering the leader sequence of the variable region. The reverse primer is located in the constant region of the heavy chain and light chain. There is no engineered restriction site in the primer.

[0382] - Reaction settings: 5x One-step RT-PCR buffer 5.0 μl, dNTP mixture (containing 10 mM of each dNTP) 0.8 μl, primer set 0.5 μl, One-step RT-PCR enzyme mixture 0.8μl, template RNA 2.0μl, RNase-free water to 20.0μl. Total volume 20.0μl. PCR conditions: reverse transcription: 50℃, 30min; initial PCR activation: 95℃, 15min; cycle: 94℃, 25sec; 54℃, 30sec; 72℃, 30sec for 20 cycles; final extension: 72℃, 10min.

[0383] - Second round of semi-nested PCR: RT-PCR products from the first round of reactions were further amplified in a second round of PCR. 12 separate heavy chain and 11 light chain RT-PCR reactions were set up using semi-nested primer sets specific for the antibody variable regions.

[0384] - Reaction setup: 2x PCR mix 10 μl; primer set 2 μl; first round PCR product 8 μl; total volume 20 μl; hybridoma antibody clone reporter PCR conditions: initial denaturation at 95°C for 5 min; 95°C for 25 sec, 57°C for 30 sec, 68°C for 30 sec for a total of 25 cycles; final extension is 10 min at 68°C.

[0385] -After PCR is complete, the PCR reaction samples are migrated on an agarose gel to visualize the amplified DNA fragments. After sequencing the DNA fragments of more than 15 clones amplified by nested RT-PCR, several mouse antibody heavy and light chains have been cloned and are correct. Protein sequence alignment and CDR analysis identified a heavy chain and a light chain. Since the amino acids at positions 26, 40, and 55 of the variable heavy chain and the amino acid at position 40 of the variable light chain are critical for binding performance, they can be restored to mouse sources. The resulting candidates are depicted below. ( Padlan1991.Mol.Immunol.28,489–498; Harris and Bajorath.1995.ProteinSci.4,306– 310 ).

[0386] Annotation of the antibody fragment sequences (SEQ ID No.: 13-22): In bold and underline are CDRs 1, 2, 3 in chronological order; in italics are constant regions; the hinge region is highlighted with bold letters, and the histidine tag is highlighted with bold and italic letters.

[0387] SEQ ID No.: 13 (AM-VH-C)

[0388]

[0389] SEQ ID No.: 14 (AM-VH1)

[0390]

[0391] SEQ ID No.: 15 (AM-VH2-E40)

[0392]

[0393] SEQ ID No.: 16 (AM-VH3-T26-E55)

[0394]

[0395] SEQ ID No.:17(AM-VH4-T26-E40-E55)

[0396]

[0397] SEQ ID No.: 18 (AM-VL-C)

[0398]

[0399] SEQ ID No.: 19 (AM-VL1)

[0400]

[0401] SEQ ID No.:20(AM-VL2-E40)

[0402]

[0403] Example 2

[0404] Immunoassay Development

[0405] An immunoassay was developed using antibodies raised against human ADM peptides (NT-H, MR-H, and CT-H; see Table 1).

[0406] Labeling procedure (tracer): 100 μg (100 μl) of antibody (1 mg / ml in PBS, pH 7.4) was mixed with 10 μl of acridine NHS ester (1 mg / mL in acetonitrile, InVent GmbH, Germany) ( EP0353971 ) and incubate at room temperature for 20 min. Purification was performed by gel filtration HPLC on SEC 400-5 (Bio-Rad Laboratories, Inc., USA). The purified labeled antibody was diluted in (300 mmol / L potassium phosphate, 100 mmol / L NaCl, 10 mmol / L Na-EDTA, 5 g / L bovine serum albumin, pH 7.0). The final concentration was approximately 800.000 relative light units (RLU) per 200 μL labeled compound (approximately 20 ng labeled antibody). Acridinium ester chemiluminescence was measured using AutoLumat LB 953 (Berthold Technologies GmbH & Co. KG).

[0407] Solid phase: Polystyrene tubes (Greiner Bio-One International AG, Austria) were coated with antibodies (1.5 μg antibody / 0.3 mL 100 mmol / L NaCl, 50 mmol / L TRIS / HCl, pH 7.8) (room temperature for 18 h). After blocking with 5% bovine serum albumin, the tubes were washed with PBS, pH 7.4 and dried under vacuum.

[0408] Calibrators: Synthetic human ADM (hADM) (Bachem, Switzerland) was diluted linearly with 50 mM Tris / HCl, 250 mM NaCl, 0.2% Triton X-100, 0.5% BSA, 20 tablets / L of complete protease inhibitor cocktail tablets (Roche AG), pH 7.8. Calibrators were stored at -20°C until use.

[0409] ADM immunoassay: 50 μl of sample (or calibrator) is drawn into the coated tube, and after adding the labeled secondary antibody (200 μl), the tube is incubated at room temperature for 2 h. Unbound tracers are removed by washing 5 times (1 ml each time) with a cleaning solution (20 mM PBS, pH 7.4, 0.1% Triton X-100). The tube-bound chemiluminescence is measured using LB 953 (Berthold Technologies GmbH & Co. KG). Antibodies are used as coated tubes and labeled antibodies in sandwich immunoassays, and are combined in the following variations (see Table 2). Incubation is carried out as described under the hADM immunoassay. The results are given as the ratio of specific signal (at 10 ng / mL ADM) / background (samples without ADM) signal.

[0410] Table 2:

[0411] Signal-to-Noise Ratio NT-ADM tracer MR-ADM tracer CT-ADM tracer NT-ADM / 195 241 MR-ADM 204 / 904 CT-ADM 260 871 /

[0412] Surprisingly, we found that the combination of MR-ADM and CT-ADM was the combination with the highest signal-to-noise ratio. Subsequently, we used this antibody combination for further studies to measure bio-ADM. We used anti-MR-ADM antibody as the solid phase antibody and anti-CT-ADM antibody as the labeling antibody. A typical dose / signal curve is shown in Figure 1 middle.

[0413] Example 3

[0414] Stability of human adrenomedullin

[0415] Human ADM was diluted in human citrated plasma (n=5, final concentration 10 ng ADM / mL) and incubated at 24° C. At selected time points, aliquots were frozen at −20° C. Upon thawing of the samples, hADM was immediately quantified using the hADM immunoassay described above.

[0416] Table 3 shows the stability of hADM in human plasma at 24°C.

[0417] Table 3:

[0418] Time (h) Average ADM recovery (N=5) Relative loss of immune responsiveness % loss of immune responsiveness / h 0 100 / / 2 99.2 0.8 0.4 4 96.4 3.6 0.8 8 88.2 11.8 1.5 Average value: 0.9% / h

[0419] Surprisingly, the pre-analytical stability of the analytes was high (mean loss of immunoreactivity was only 0.9% / h) using the antibody combinations MR-ADM and CT-ADM in a sandwich immunoassay. In contrast, using other assays, the reported plasma half-life was only 22 min ( Hinson et al., 2000 Endocrine Reviews21(2):138-167Since the time from sampling to analysis is less than 2 h in daily hospital practice, the ADM detection method used is suitable for routine diagnosis. Remarkably, no unconventional additives (such as aprotinin ( Ohta et al., 1999. Clin Chem 45(2): 244-251 )).

[0420] Example 4

[0421] Reproducibility of calibrator preparations and inter-preparation variation of calibrators

[0422] We found high variation in the results of the preparation of calibrators for the ADM assay (average CV 8.5%, see Table 4). This is probably due to the high adsorption of hADM to plastic and glass surfaces ( Lewis et al., 1998. Clinical Chemistry 44(3): 571-577 ). This effect was only slightly reduced by the addition of detergents (up to 1% Triton X 100 or 1% Tween 20), proteins (up to 5% BSA) and high ionic strength (up to 1 M NaCl) or a combination thereof. Surprisingly, the recovery and reproducibility of the ADM assay calibrator preparations improved significantly, reaching an inter-preparation CV of <1%, if an excess of anti-NT-ADM antibody (10 μg / mL) was added to the calibrator dilution buffer (Table 4). The coefficient of variation is given from 5 independent preparation runs. The calibrators were measured using the ADM assay described above (s / nr = signal-to-noise ratio). For all subsequent studies, we used an ADM assay based on calibrators prepared in the presence of 10 μg / mL NT-ADM antibody as a supplement and 10 μg / mL NT-ADM antibody in the tracer buffer.

[0423] Fortunately, the presence of the N-terminal antibody did not affect the bio-ADM signal generated by the combination of MR- and C-terminal antibodies ( Figure 1 ).

[0424] Table 4:

[0425]

[0426] Example 5

[0427] Sensitivity

[0428] The goal of the assay sensitivity is to fully cover ADM concentrations in healthy subjects and concentrations significantly lower.

[0429] Bio-ADM concentrations in healthy subjects

[0430] Healthy subjects (n=88, 57 females, 31 males, mean age: 42.2 years) were measured using the bio-ADM assay ( Weber et al., 2017. JALM, 2(2): 222-233 ). The median interquartile range (IQR) was 13.7 (9.6–18.7) pg / mL and the mean (SD) was 15.6 (9.2) pg / mL. Since the assay sensitivity (detection limit) was 3 pg / mL, 100% of healthy subjects could be detected using the bio-ADM assay.

[0431] Example 6

[0432] Study Design and Populations of the MPP

[0433] Malmö Prevention Project The MPP was funded in the mid-1970s to explore CV risk factors in the general population and recruited 33,346 individuals living in Malmö ( Fedorowski et al., 2010. Eur Heart J 31:85–91 Between 2002 and 2006, a total of 18,240 original participants responded to the invitation (participation rate 70.5%) and underwent screening, which included a comprehensive physical examination and blood sample collection ( Fava et al. 2013.Hypertension2013;61:319–26 ). In this study, the retest in the MPP was considered as the baseline. Subjects who already had CVD at baseline were excluded. In 4,364 patients, Bio-ADM was measured from plasma collected during the baseline assessment. At the time of blood sampling, 34 patients had been diagnosed with Alzheimer's disease. In the subsequent 7 years, 187 patients developed AD (developed AD). Informed consent was obtained from all participants, and the Ethical Committee of Lund University (Lund, Sweden) approved the study protocol.

[0434] Information on dementia diagnoses was sought from the Swedish National Patient Register (SNPR). Diagnoses in the register were collected according to different revisions of the International Classification of Diseases (ICD) codes 290, 293 (ICD-8), 290, 331 (ICD-9) or F00, F01, F03, G30 (ICD-10). Since 1987, the SNPR includes all inpatient care in Sweden and in addition contains outpatient data, including day surgery and psychiatric care from both private and public caregivers after 2000. Dementia of all causes was diagnosed according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders (DSM)-III revision, while DSM-IV criteria were used for Alzheimer's disease and vascular dementia. Diagnosis was verified by a comprehensive review of medical records and available neuroimaging data. The study physician assigned a final diagnosis to each patient and consulted a geriatrician specializing in cognitive impairment in unresolved cases. We selected a dataset for a case-control study and measured MR-proADM in a sub-cohort of the MPP (n=250 controls, and n=150 subjects with incident AD). In addition, bio-ADM was measured in an independent cohort of patients who had been diagnosed with Alzheimer's disease at the time of blood sampling (=current AD; n=150).

[0435] MR-proADM in plasma was measured using a commercial fully automated homogeneous time-resolved fluorescence immunoassay (BRAHMS MR-proADM KRYPTOR; BRAHMS GmbH, Hennigsdorf, Germany) ( Caruhel et al., 2009. Clin Biochem. 42(7-8):725-8 ).

[0436] Statistical analysis:Values ​​are expressed as mean and standard deviation, median and interquartile range (IQR) or appropriate counts and percentages. Comparison of groups of continuous variables was performed using the Kruskal-Wallis test. Biomarker data were logarithmically transformed. In univariate and multivariate analyses, Cox proportional hazard regression was used to analyze the effect of risk factors on survival. The assumption of proportional hazards was tested for all variables. For continuous variables, the hazard ratio (HR) was standardized to describe the HR of biomarker changes of one IQR. The significance level of the 95% confidence interval (CI) and chi-square (Wald test) of the risk factor was given. The predictive value of each model was assessed by the model likelihood ratio chi-square statistic. The consistency index (C index) was given as a measure of effect. It is equivalent to the concept of AUC adopted for binary results. For multivariate models, a bootstrap-corrected version of the C index was given. Survival curves drawn by the Kaplan-Meier method were used for illustrative purposes. In order to test the independence between bio-ADM and clinical variables, we used the likelihood ratio chi-square test for the nested model.

[0437] All statistical tests were two-tailed, and a two-sided p-value of 0.05 was considered significant. http: / / www.r-project.org , library Design, Hmisc, ROCR) and Statistical Package for the Social Sciences (SPSS) version 22.0 (SPSS Inc., Chicago, Illinois, USA) were used for statistical analysis.

[0438] result:

[0439] Baseline characteristics of the cohort are shown in Table 5.

[0440] Table 5:

[0441]

[0442]

[0443] The bio-ADM concentrations in the MPP cohort and the independent Alzheimer's disease cohort are shown in Figure 2 The bio-ADM concentrations in patients who developed AD over time (incident AD) and in an independent cohort with AD at the time of blood sampling (current AD) were significantly lower compared to the non-AD group (p=0.01 and <0.0001, respectively). The current AD group (n=34) from the MPP cohort also showed lower bio-ADM concentrations compared to the non-AD group, but this was not statistically significant due to the small sample size.

[0444] Low bio-ADM plasma concentrations strongly predicted Alzheimer's disease with a hazard ratio (HR) of 0.73 (CI 0.6–0.87; p<0.001). Figure 3 Kaplan-Meier plots for prediction of Alzheimer's disease using bio-ADM concentrations are shown (current AD cases were excluded from the analysis). The lowest quartile was associated with the highest risk of developing AD.

[0445] We selected 400 patients from the MPP cohort (without prior CVD and AD) to generate a case-control dataset, with n=250 subjects who did not develop AD and n=150 subjects who developed AD during the 7-year follow-up period. Likewise, bio-ADM concentrations were significantly lower in patients who developed AD and in patients with current AD (independent cohorts) compared to the non-AD group (p<0.0001 for all comparisons) ( Figure 4 In addition, MR-proADM was measured in MPP case-control subjects and was slightly but not significantly higher in the AD-developing group compared with the AD-free group ( Figure 5 ).

[0446] Next, we combined the two biomarkers, bio-ADM and MR-proADM. In order to calculate the ratio, the concentrations of the two markers must preferably be expressed in the same unit (e.g., pmol / L). Therefore, from the perspective of calculating the ratio, the concentration of bio-ADM is calculated in pmol / L. When compared to subjects without AD, the ratio of bio-ADM to MR-proADM was significantly reduced in subjects with the development of AD (p<0.0001; Figure 6 ). Bio-ADM alone strongly predicted Alzheimer's disease with an odds ratio (OR) of 0.44 (CI 0.33–0.58). However, the ratio of bio-ADM to MR-proADM was better than bio-ADM alone (p<0.001) with an OR of 0.32 (CI 0.23-0.44). The corresponding receiver operating characteristic curves (ROC plots) for bio-ADM and the ratio of bio-ADM to MR-proADM are shown in Fig. 7A and Figure 7B The results showed that the AUC of bio-ADM was 0.67 (95% CI 0.61-0.72) and the AUC of the ratio between bio-ADM and MR-proADM was 0.73 (95% CI 0.68-0.78). In addition, both bio-ADM and the ratio of bio-ADM to MR-proADM were independent of age and gender.

[0447] Example 7 - Administration of NT-H in Healthy Humans

[0448] The study was conducted as a randomized, double-blind, placebo-controlled study in healthy male subjects using single ascending doses of NT-H antibody (first group, 0.5 mg / kg; second group, 2 mg / kg; third group 8 mg / kg) administered as intravenous (iv) infusion in 3 consecutive groups of 8 healthy male subjects each (n=6 for active agent and n=2 for placebo per group).

[0449] The main inclusion criteria were written informed consent, age between 18 and 35 years, agreement to use reliable contraceptive methods, and BMI between 18 and 30 kg / m 2 between.

[0450] Subjects in the study unit received a single iv dose of NT-H antibody (0.5 mg / kg; 2 mg / kg; 8 mg / kg) or placebo by slow infusion over a 1 hour period.

[0451] There was no difference in baseline bio-ADM values ​​among the 4 groups. The median bio-ADM value was 7.1 pg / mL in the placebo group, 6.8 pg / mL in the first treatment group (0.5 mg / kg), 5.5 pg / mL in the second treatment group (2 mg / kg), and 7.1 pg / mL in the third treatment group (8 mg / mL).

[0452] The results showed that in healthy human subjects, bio-ADM values ​​increased rapidly within the first 1.5 hours after NT-H antibody administration, then reached a plateau and slowly decreased ( Figure 8 ).

Claims

1. Use of an anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or an anti-ADM non-Ig backbone in the preparation of a medicament for preventing and treating dementia in a subject, wherein the anti-ADM antibody or anti-ADM fragment or anti-ADM non-Ig backbone binds to the N-terminal portion (aa 1-21) of adrenomedullin: YRQSMNNFQGLRSFGCRFGTC (SEQ ID No.: 21).

2. Use of the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig skeleton according to claim 1 in the preparation of a drug for preventing and treating dementia in a subject, wherein the level of mature ADM-NH2 shown in SEQ ID No.: 4 determined in the subject's body fluid sample of the subject is lower than a threshold level, and / or the subject has a marker level ratio, which is the ratio of the level of mature ADM-NH2 shown in SEQ ID No.: 4 determined in the subject's body fluid sample to the level of adrenomedullin precursor or its fragment determined in the subject's body fluid sample, and wherein the marker level ratio is lower than the ratio threshold.

3. Use of the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig backbone according to claim 2 in the preparation of a medicament for preventing and treating dementia in a subject, wherein the fragment of the adrenomedullin precursor is selected from PAMP (SEQ ID No.: 2), MR-proADM (SEQ ID No.: 3), ADM-Gly (SEQ ID No.: 5) and CT-proADM (SEQ ID No.: 6).

4. Use of the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig backbone according to claim 2 or 3 in the preparation of a medicament for preventing and treating dementia in a subject, wherein the threshold level of mature ADM-NH2 shown in SEQ ID No.: 4 is equal to or lower than 15 pg / mL.

5. Use of the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig backbone according to claim 4 in the preparation of a medicament for preventing and treating dementia in a subject, wherein the threshold level of mature ADM-NH2 shown in SEQ ID No.: 4 is equal to or lower than 10 pg / mL.

6. Use of the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig backbone according to claim 5 in the preparation of a medicament for preventing and treating dementia in a subject, wherein the threshold level of mature ADM-NH2 shown in SEQ ID No.: 4 is equal to or lower than 5 pg / mL.

7. Use of the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig backbone according to claim 2 or 3 in the preparation of a medicament for preventing and treating dementia in a subject, wherein the marker level ratio is in the range of 0.2 to 0.

75.

8. Use of the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig backbone according to claim 7 in the preparation of a medicament for preventing and treating dementia in a subject, wherein the marker level ratio is in the range of 0.3 to 0.

6.

9. Use of the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig backbone according to claim 8 in the preparation of a medicament for preventing and treating dementia in a subject, wherein the marker level ratio is in the range of 0.4 to 0.5.

Images