Modified and unmodified peptidyl-glycine alpha-amidating monooxygenase (PAM) for use in the treatment or prevention of cerebral hypoperfusion for the improvement of cerebral blood flow (CBF)

Abstract

The invention relates to modified or unmodified Peptidylglycine a-amidating monooxygenase (PAM) or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of cerebral blood flow (CBF), as well as to corresponding pharmaceutical formulations and kits.

Description

[0001] Modified and unmodified Peptidyl-glycine alpha-amidating monooxygenase (PAM) for use in the treatment or prevention of cerebral hypoperfusion for the improvement of cerebral blood flow (CBF).

[0002] The present invention relates to modified or unmodified Peptidylglycine alpha-amidating monooxygenase (PAM) or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of cerebral blood flow (CBF). The present invention is furthermore directed to corresponding pharmaceutical formulations of modified or unmodified PAM for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF.

[0003] Background of the invention

[0004] Peptidyl-glycine alpha-amidating monooxygenase (PAM) is the only known enzyme for the conversion of inactive peptide hormones into their active form via C-terminal alpha amidation (Eipper et al., Annu Rev Neurosci 1992, 15: 57-85). More than half of known peptide hormones require this process for their activation. PAM specifically recognizes C-terminal glycine residues in its substrates, cleaves glyoxylate from the peptide’s C-terminal glycine residue in a two-step enzymatic reaction leading to the formation of C-terminally alpha-amidated peptide hormones, wherein the resulting alpha-amide group originates from the cleaved C-terminal glycine (Prigge et al. 2004. Science 304(5672): 864–67). This amidation reaction takes place in the lumen of secretory granules prior to exocytosis of the amidated product (Martinez and Treston 1996. Molecular and Cellular Endocrinol 123: 113–17). Alpha-amidated peptides are for example adrenomedullin (ADM), substance P, vasopressin, neuropeptide Y, Amylin, calcitonin, neurokinin A and others. However, previously it was demonstrated that PAM can also catalyze the formation of alpha-amides from glycinated substrates of non-peptide character, e.g. N-fatty acyl-glycines, which are converted by PAM to primary fatty acid amides (PF AMs) like oleamide. The identified and purified peptidyl-glycine amidating activities were shown to be dependent on copper and ascorbate (Emeson et al. 1984. Journal of Neuroscience: 2604–13; Kumar et al. 2016. J Mol Endocrinol 56(4):T63-76; Wand et al. 1985. Neuroendocrinology 41: 482–89).

[0005] In humans, the PAM gene is located at chromosome 5q21.1 having a length of 160 kb containing 25 known exons (Gaier et al. 2014. BMC Endocrine Disorders 14}. At least 6 isoforms are known to be generated by alternative splicing (SEQ ID Nos. 1-6; SEQ ID Nos. 30 - 35 show the respective prepro-forms). The PAM enzyme was found to be expressed at different levels in almost all mammalian cell types, with significant expression in airway epithelium, endothelial cells, ependymal cells in the brain, adult atrium, brain, kidney, pituitary, gastrointestinal tract and reproductive tissues (Chen et al. 2018. Diabetes Obes Metab 20 Suppl 2:64-76; Oldham et al. 1992. Biochem Biophys Res Commun 184(1): 323–29; Schafer et al. 1992. J Neurosci 12(1): 222–34).

[0006] The precursor protein (1-973 amino acids) of the largest known PAM Isoform 1 (SEQ ID No.

[0007] 30) encoded by the PAM cDNA is depicted in Figure 1. The N-terminal signal sequence (amino acids 1-20) assures direction of the nascent PAM polypeptide into the secretory lumen of endoplasmic reticulum and is subsequently cleaved co-translationally. Afterwards the PAM-pro-peptide is processed by the same machinery used for the biosynthesis of integral membrane proteins and secreted proteins including cleavage of the pro-region (amino acids 21-30), assuring proper folding, disulfide bond formation, phosphorylation and glycosylation (Bousquet-Moore et al. 2010. J Neurosci Res 88(12):2535-45). The sequences of PAM isoforms 1 to 6 after cleavage of the N-terminal signal sequence (amino acid 1-20) as well as the proregion (amino acids 21 to 30) are given as SEQ ID No. 1 to 6.

[0008] As depicted in Figure 1, the PAM cDNA further encodes two distinct enzymatic activities. The first enzymatic activity is named peptidyl-glycine alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3), is an enzyme, capable of catalyzing the conversion of a C-terminal glycine residue to an alpha hydroxy-glycine. The second activity is named peptidyl-a-hydroxy-glycine alpha-amidating lyase (PAL; EC 4.3.2.5) is an enzyme capable of catalyzing the conversion of an alpha hydroxy-glycine to an alpha-amide with subsequent glyoxylate release. The sequential action of these separate enzymatic activities results in the overall peptidyl-glycine alpha amidating activity. The first enzymatic activity (PHM) is located directly upstream of the proregion (within amino acids 31-494 of isoform 1 (SEQ ID No. 7)). The second catalytic activity (PAL) is located after exon 16 in isoform 1 within amino acids 495-817 (SEQ ID No. 8). As depicted in Figure 1, both activities may be encoded together within of one polypeptide as a membrane-bound protein (isoforms 1, 2, 5, 6; corresponding to SEQ ID No. 1, 2, 5 and 6) as well within of one polypeptide as a soluble protein lacking the transmembrane domain (TMD) (isoforms 3 and 4; corresponding to SEQ ID No. 3 and 4). While isoforms 1, 2, 5 and 6 remain in the outer plasma membrane after fusion of secretory vesicles with the plasma membrane with subsequent endocytosis and recycling or degradation, soluble PAM isoforms lacking the TMD (isoforms 3 and 4) (amino acids 864-887) are co-secreted with the peptide-hormones (Wand et al. 1985 Metabolism 34(11): 1044–52). Furthermore, prohormone convertases may convert membrane bound PAM protein into soluble PAM protein by cleavage within the flexible region (exons 25 / 26) connecting PAL with the TMD during the secretory pathway (Bousquet-Moore et al. 2010. J Neurosci Res 88(12):2535-45). The PHM subunit may be cleaved from soluble or membrane bound PAM within the secretory pathway by prohormone convertases that address a double-basic cleavage-site in the exon 16 region. Furthermore, during endocytosis the full-length PAM protein may be also converted into a soluble form due to the action of alpha- and gamma secretases (Bousquet-Moore et al. 2010. J Neurosci Res 88(12):2535-45). Membrane bound PAM from late endosome can be further secreted in form of exosomal vesicles.

[0009] PHM and PAL activities, as well as the activity of the full-length PAM were determined in several human tissues and body fluids. However, the separated PHM and PAL activities in soluble forms will also lead to formation of C-terminally alpha amidated products from C-terminally glycinated substrates when allowed to perform their separate reactions in the same compartment, body-fluid or in vitro experimental setup. How the transfer of the PHM hydroxylated product to the PAL takes place is not exactly understood to date. There is evidence that the hydroxylated product is released into solution and is not directly transferred from PHM to PAL (Yin et al. 2011. PLoS One 6(12):e28679). Additionally, the source of PAM in circulation remained unclear.

[0010] The partial reaction of PHM is depicted in Figure 2. PHM is a copper dependent monooxygenase responsible for stereo-specific hydroxylation of the C-terminal glycine at the alpha carbon atom. During the hydroxylation reaction ascorbate is believed to be the naturally occurring reducing agent, while the oxygen in the newly formed hydroxyl group was shown to originate from molecular oxygen. The partial reaction of the PAL is depicted in Figure 2. The catalytic action of PAL involves proton abstraction form the PHM-formed hydroxy-glycine by a protein-backbone derived base and a nucleophilic attack of hydroxyl-group oxygen to the divalent metal leading to a cleavage of glyoxylate and formation of a C-terminal amide.

[0011] One example of a substrate of PAM is Adrenomedullin-Gly (SEQ ID No. 14). The Adrenomedullin (ADM) mRNA encodes a preprohormone of 185 amino acids (SEQ ID No.

[0012] 11), the pre-pro- Adrenomedullin that is enzymatically converted into Proadrenomedullin by cleavage of the N-terminal signal peptide. Proadrenomedullin is then further processed by several prohormone convertases to result in four peptides, namely

[0013] PAMP-Gly (SEQ ID No. 12): Proadrenomedullin amino-terminal peptide or Proadrenomedullin N-terminal 20 peptide with a C-terminal glycine residue MR-proADM (SEQ ID No. 16): Mid-regional Proadrenomedullin, a stable and inert peptide

[0014] ADM-Gly (SEQ ID No. 14): C-terminally glycine extended, inactive precursor of biologically active ADM (bio-ADM (SEQ ID No. 15))

[0015] CT-proADM (SEQ ID No. 13): C-terminal Proadrenomedullin or Adrenotensin.

[0016] Cerebral blood flow (CBF) is fundamentally essential for the maintenance of brain function, providing the necessary nutrients and oxygen while removing metabolic waste.

[0017] It has been shown that CBF changes significantly in a variety of medical conditions, including neuroinflammation and neurodegeneration diseases (e.g. Alzheimer’s (AD), Parkinson's (PD), Vascular Dementia (VD), Multiple Sclerosis (MS), Huntington's Disease (HD), Amyloid Lateral Sclerosis (ALS)), cardiovascular diseases (e.g. stroke, heart failure, atrial fibrillation, carotid artery stenosis, hypotension, hypertension), metabolic disorders (e.g. Diabetes Mellitus (DM)) and many others (Mokhber et al. 2021. The Neuroradiology Journal, Vol. 34(4): 300-307).

[0018] In the aging population, a notable reduction in CBF is observed, particularly in the cortex regions such as the lateral occipital, cingulate, precuneus, temporal, parietal, insular, and frontal lobes, primarily measured using Arterial Spin Labeling MRI (ASL-MRI), supplemented by PET and SPECT imaging techniques (Chen et al. 2011. Neuroimage. 55(2): 468-478; Leenders et al.1990. Brain 113 (Pt 1): 27-47; Claus et al. 1998. Neurobiol Aging 19(1): 57-64). The gradual decline in cerebral blood flow with aging is estimated within a range of 2-5% per year (“chronic age-related cerebral hypoperfusion“), but can be severely accelerated in pathological conditions („pathology-induced cerebral hypoperfusion“) (Graff et al. 2022. Cerebrovasc Dis.

[0019] 52(1): 11-20).

[0020] The role or influence of PAM in some clinical conditions has been described. The presence of alpha-amidating activity in human circulation was initially proved by Wand et al. (Wand et al.

[0021] 1985. Neuroendocrinology 41, 482-489). They reported no sex differences but some variations of PAM activity in certain disease states: Plasma PAM activities were increased in hypothyroid adults as well as in patients with medullary thyroid carcinoma. The activity of PAM in tissues of medullary thyroid carcinoma, pheochromocytoma and pancreatic islet tumors were shown to be elevated suggesting increased formation of amidated peptides in endocrine tumor tissues (Gether et al. 1991. Mol. Cell. Endocrinol. 79, 53-63; Wand et al. 1985. Neuroendocrinology 41, 482-489}. Patients suffering from multiple endocrine neoplasia type 1 (MEN-1) and pernicious anemia showed a decreased plasma PAM activity in comparison to healthy control subjects. The presence of amidating activity in human cerebrospinal fluid (CSF) was shown by Wand and colleagues. In patients suffering from Alzheimer’s disease (AD) plasma PAM activities were shown to be unaltered when compared to healthy controls, while CSF PAM activities were significantly decreased in comparison to activities from normal specimen (Wand et al. 1987. Neurology 37, 1057-1061}. In addition, in WO2015 / 103594 it was proposed that the presence of PAM-Protein in CSF detected by mass spectrometry of AD-patients was reduced compared to healthy controls. Moreover, ADM, one of the hormones amidated by PAM, was shown to be reduced in patients with prevalent and incident Alzheimer’s disease (WO2019 / 154900). Direct associations of circulating PAM activities were reported being associated with prediction, diagnosis or progression of AD in W02021 / 170816A1 and WO2021 / 170752A1. PAM activities of patients suffering from multiple sclerosis (MS) were shown to be increased in CSF, with a significant decrease in serum (Tsukamoto et al. 1995. Intern. Med. 34, 229-232 and W02010 / 005387}. An association between plasma activity of PAM and type-2-diabetes was described in (WO2014 / 118634}. Further, WO2021170816A1 and WO2021170752A1 show an increase of PAM activity in circulation due to acute conditions such as sepsis and shock.

[0022] However, nothing is known about PAM for the treatment or prevention of cerebral hypoperfusion and the influence of PAM on cerebral blood flow. Detailed description of the invention

[0023] The examples in the description of the present invention show that injection of Peptidylglycine alpha-amidating monooxygenase (PAM), and in particular modified PAM (e.g. by PEGylation) significantly increased hemodynamics, such as cerebral blood flow in rats with cerebral hypoperfusion (Example 7). Furthermore, it has been demonstrated that PAM, and in particular modified PAM (e.g. by PEGylation) had a protective effect on brain blood vessels in both infarct-affected and adjacent brain areas following ischemia (Example 8). This clearly demonstrates that PAM, and in particular modified PAM (e.g. by PEGylation) is able to prevent cerebral hypoperfusion in a subject in need thereof and to improve or maintain cerebral blood flow.

[0024] It is the surprising finding of the present invention that PAM, and in particular modified PAM (e.g. by PEGylation) is suitable for use in therapy or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF.

[0025] Subject matter of the present invention is modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF.

[0026] In the present invention “Treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF” likewise relates to “treatment or prevention of cerebral hypoperfusion in a subject in need thereof whereby the CBF is improved”.

[0027] Certain embodiments of the invention relate to modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of cerebral blood, wherein said subject has a reduction in CBF. The term “cerebral hypoperfusion” refers in particular to a condition characterized by an insufficient supply of blood to the brain, which is below the necessary level to maintain optimal cellular function and metabolic processes. „Cerebral hypoperfusion“ particularly comprises „chronic age-related cerebral hypoperfusion“ and „pathology -induced cerebral hypoperfusion“.

[0028] In this regard, “chronic age-related cerebral hypoperfusion” refers to a persistent and progressive reduction in cerebral blood flow that is commonly associated with the aging process. Chronic hypoperfusion in the brain can lead to a sustained state of reduced oxygen and nutrient supply to neural tissues. Overtime, this condition may contribute to neurodegenerative changes, cognitive decline, and an increased risk of developing conditions such as vascular dementia and Alzheimer's disease.

[0029] The pathophysiological mechanisms underlying chronic age-related cerebral hypoperfusion may involve endothelial dysfunction, arteriosclerosis, and microvascular rarefaction, which collectively impair the brain's vascular autoregulation and perfusion efficiency.

[0030] Moreover, “pathology-induced cerebral hypoperfusion” refers to a reduction in cerebral blood flow that is caused by an underlying pathological condition. Unlike age-related hypoperfusion, pathology-induced cerebral hypoperfusion can occur at any age and is typically associated with specific diseases or medical conditions. These conditions may include, but are not limited to, stroke, traumatic brain injury, cardiac arrest, severe hypotension, and various forms of cardiovascular disease. The resultant hypoperfusion can lead to insufficient delivery of oxygen and nutrients to brain tissues, potentially causing ischemic injury, neuronal death, and subsequent neurological deficits.

[0031] As used herein, cerebral hypoperfusion is characterized by a reduction in CBF; accordingly, prevention or treatment of cerebral hypoperfusion inherently includes maintaining or improving CBF.

[0032] Certain embodiments of the invention relate to modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF, wherein the CBF is defined as volume of blood expressed in millilitres that passes through 100 g of brain tissue per minute.

[0033] The term “cerebral blood flow” as used in the context of the present invention is defined as the blood volume that flows per unit mass per unit time in brain tissue and is typically expressed in units of ml blood / (100g tissue * min). Alternatively, one may express CBF in terms of flow per unit volume of brain tissue, thus in ml blood / (100ml tissue * min). Therein, CBF, as used in the context of the present invention, refers to the global cerebral blood flow. If not stated otherwise, the CBF in the context of the present invention is expressed in units of ml blood / (100g tissue * min).

[0034] The global cerebral blood flow is an aggregate measure that averages the blood flow rates of both grey matter and white matter. This value provides an overall assessment of the brain's perfusion status and is crucial for understanding the brain's metabolic needs and detecting abnormalities in blood flow that may indicate pathological conditions. The normal average of global CBF in adults is about 50 ml / (100g * min) (Lassen, 1985. J. Cereb. Blood Flow Metab. 5, 347 349}, with lower values in the white matter [~20ml / (100g * min)] and greater values in the grey matter [~80ml / (100g * min)] (Vavilala et al. 2002. Anesthesia!. Clin. N. Am. 20, 247 264}.

[0035] The global CBF is the mean CBF of grey matter and white matter CBF values. In the context of the present invention the terms “CBF” and “global CBF” are used interchangeably, unless explicitly specified otherwise.

[0036] Certain embodiments of the invention relate to modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF, wherein the cerebral blood flow in said subject is below a certain threshold.

[0037] Certain embodiments of the invention relate to modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in said subject in need thereof for the improvement of CBF, wherein the cerebral blood flow in said subject is below a certain threshold and wherein said threshold is defined as a reduction of the CBF below 50 mL / 100g (of brain tissue) per min, preferably below 30 mL / 100g (of brain tissue) per min, more preferably below 15 mL / 100g (of brain tissue) per min and most preferably below 10 mL / 100g (of brain tissue) per min.

[0038] In certain embodiments of the invention said subject is characterized by a level of PAM and / or its isoforms and / or fragments thereof below a certain threshold in a bodily fluid of said subject. In particular embodiments, the level of PAM and / or its isoforms and / or fragments thereof is detected as total PAM concentration and / or total PAM activity.

[0039] More particularly this means the total concentration of all PAM isoforms, and / or the total alpha-amidating activity determinably in a sample obtained from said subject.

[0040] In certain embodiments, particular thresholds for total PAM concentration in a sample of bodily fluid, obtained from said subject are equal or below 75 ng / mL, preferably equal or below 65 ng / mL, more preferably equal or below 55 ng / mL, more preferably equal or below 45 ng / mL, more preferably equal or below 35 ng / mL and most preferably equal or below 30 ng / mL. In certain embodiments, particular thresholds for PAM activity in a sample of bodily fluid,, obtained from said subject are equal or below 11.5 μg / L*h, preferably equal or below 10.5 μg / L*h, more preferably equal or below 9.5 μg / L*h, more preferably equal or below 8.5 μg / L*h, more preferably equal or below 7.5 μg / L*h and most preferably equal or below 6.5 μg / L*h, when PAM activity is measured in serum, wherein the quantification of activity of PAM in Units is as described in Example 4 and the term Units / L defines the activity of PAM in Units per 1 L of sample material.

[0041] In certain embodiments, particular thresholds for PAM activity in a sample of bodily fluid,, obtained from said subject are equal or below 16.9 μg / L*h, preferably equal or below 15.4 μg / L*h, more preferably equal or below 13.9 μg / L*h, more preferably equal or below 12.3 μg / L*h, more preferably equal or below 10.9 μg / L*h and most preferably equal or below 9.4 μg / L*h, when PAM activity is measured in Li-Heparin, wherein the quantification of activity of PAM in Units is as described in Example 4 and the term Units / L defines the activity of PAM in Units per 1 L of sample material.

[0042] The person skilled in the art is aware that the activity may also be determined in other bodily fluids and how to determine the respective threshold.

[0043] In certain embodiments of the invention said sample of bodily fluid of said subject is selected from the group of whole blood, serum or plasma.

[0044] The levels of PAM or fragments thereof of the present invention have been determined with the described assays as outlined in Kaufmann et al. 2021 and Example 4 for the measuring of amidating activity and as outlined in Ilina et al. 2023 for the measuring of PAM concentration.

[0045] The level of PAM or fragments thereof may be the total PAM concentration or PAM activity, respectively. The mentioned threshold values above might be different in other assays, if these have been calibrated differently from the assay systems used in the present invention. Therefore, the mentioned cut-off values above shall apply for such differently calibrated assays accordingly, taking into account the differences in calibration. One possibility of quantifying the difference in calibration is a method comparison analysis (correlation) of the assay in question with the respective biomarker assay used in the present invention by measuring the respective biomarker (e.g., PAM or fragments thereof) in samples using both methods. Another possibility is to determine with the assay in question, given this test has sufficient analytical sensitivity, the median biomarker level of a representative normal population, compare results with the median biomarker levels as described in the literature and recalculate the calibration based on the difference obtained by this comparison. With the calibration used in the present invention, samples from 4106 normal (healthy) subjects (Swedish single-center prospective population-based Study (MPP-RES)) have been measured: median (interquartile range (IQR)) plasma PAM concentration was 78.6 ng / ml (66.4 ng / ml -92.5 ng / ml).

[0046] With the calibration used in the present invention, samples from 4942 normal (healthy) subjects (Swedish single-center prospective population-based Study (MPP-RES)) have been measured: median (interquartile range (IQR)) PAM activity in serum was 12.5 μg / (L*h (10.9 – 14.5). One Unit = one μg / (L*h).

[0047] Threshold levels can be obtained for instance from a Kaplan-Meier analysis, where the occurrence of a disease is correlated with the quartiles of the biomarker in the population. According to this analysis, subjects with biomarker levels below the 25th percentile have a significantly increased risk for getting the diseases according to the invention. This result is further supported by Cox regression analysis with full adjustment for classical risk factors: The lowest quartile versus all other subjects is highly significantly associated with increased risk for getting a disease according to the invention.

[0048] Other preferred threshold values are for instance the 20th, 10th or 5th percentile of a normal population. By using a lower percentile than the 25th percentile, one reduces the number of false positive subjects identified, but one might miss identifying subjects, who are at moderate, albeit still increased risk. Thus, one might adopt the cut-off value depending on whether it is considered more appropriate to identify most of the subjects at risk at the expense of also identifying "false positives", or whether it is considered more appropriate to identify mainly the subjects at high risk at the expense of missing several subjects at moderate risk.

[0049] In certain embodiments of the invention the CBF after treatment of the subject is increased compared to the CBF level before treatment of the subject (baseline CBF level) by at least 5% preferably by at least 10%, more preferred by at least 15%, even more preferred by at least 20%, even more preferred by at least 25%, even more preferred by at least 30%, most preferred by at least 35%.

[0050] Particularly, baseline CBF level is measured 7 days to 1 hour before treatment of the subject. Particularly, CBF level after treatment of the subject is measured 12 hours to 7 days, preferably 18 hours to 5 days, most preferred 24 hours to 72 hours if said subject is suffering from or has a risk of suffering from a disease or medical condition selected from the group comprising cardiovascular diseases or conditions, particularly selected from the group comprising atherosclerosis, hypertension, heart failure (including acute and acute decompensated heart failure), atrial fibrillation, cardiovascular ischemia, cerebral ischemic injury, cardiogenic shock, carotid artery stenosis, hypotension, hypertension, peripheral artery disease, coronary artery disease, stroke (including ischemic and haemorrhagic stroke and transient ischemic attack) and myocardial infarction, or

[0051] infections caused by infectious organisms such as bacteria, viruses, fungi or parasites, particularly selected from the group comprising SIRS, sepsis, and septic shock.

[0052] Particularly, CBF level after treatment of the subject is measured 1 -12 months, preferably 2 -9 months, more preferably 3 - 6 months, most preferred 3 months if said subject is suffering from or has a risk of suffering from a disease or medical condition selected from the group comprising

[0053] neuroinflammatory and neurodegenerative diseases; particularly dementia selected from the group comprising mild cognitive impairment (MCI), Alzheimer’s disease, vascular dementia, mixed Alzheimer’s disease, and vascular dementia, Lewy body dementia, frontotemporal dementia, focal dementias (including progressive aphasia), subcortical dementias (including Parkinson’s disease), secondary causes of dementia syndrome (including intracranial lesions, multiple sclerosis, Huntington’s Disease, Amyloid Lateral Sclerosis, Traumatic Brain Injury, Creutzfeldt-Jakob Disease); metabolic disorders, particularly diabetes mellitus type 1, diabetes mellitus type 2, obesity, and metabolic syndrome;

[0054] respiratory disorders, particularly (chronic obstructive pulmonary disease, sleep apnea) psychiatric conditions (major depressive disorder, bipolar disorder).

[0055] In certain embodiments of the invention the CBF level after treatment for the prevention of a reduction of CBF is monitored in said subject. Said monitoring is performed every 1 to 12 months, preferably every 2 to 9 months, preferably every 3 to 6 months, most preferred every 3 months.

[0056] Certain embodiments of the invention relate to modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF, wherein said CBF is assessed with a method selected from the group comprising ultrasound-based techniques for CBF measurements (Transcranial Doppler Ultrasound (TCD); Contrast-Enhanced Ultrasound (CEU), Functional Ultrasound (fUS), nuclear medicine methods (Single Photon Emission Computed Tomography (SPECT), Positron Emission Tomography (PET)), magnetic resonance imaging (MRI) techniques (Arterial Spin Labeling (ASL), phase-contrast MRI (e.g. Dynamic Susceptibility Contrast (DSC) MRI, Dynamic Contrast-Enhanced MRI (DCE-MRI)), Blood Oxygenation Level-Dependent (BOLD) Functional MRI (fMRI)) and X-ray computed tomography methods (X-Ray Computed Tomography (CT) Perfusion, Xenon-Enhanced CT (Xe-CT)).

[0057] In certain embodiments of the invention, the CBF level is assessed with a method selected from the group of ASL-MRI and / or phase-Contrast MRI and / or SPECT.

[0058] Said methods for the measurement or assessment of cerebral blood flow are described in more detail below:

[0059] • Ultrasound-based techniques

[0060] o Transcranial Doppler (TCD) Ultrasound: This technique employs Doppler ultrasound to measure the velocity of blood flow through the brain's major arteries. TCD is instrumental in monitoring cerebral hemodynamics, detecting vascular abnormalities, and assessing the cerebrovascular impact of various medical conditions and therapeutic interventions (Aaslid R et al. 1982. J Neurosurg, 57(6):769-74)

[0061] o Contrast-Enhanced Ultrasound (CEU): Utilizes microbubble contrast agents that enhance ultrasound signals to measure changes in cerebral microvascular blood volume and red blood cell velocity. This method enables real-time imaging and quantification of cerebral blood flow (Rim et al. 2001. Circulation 104, 2582- 2587; Premilovac et al. 2020. Journal of Cerebral Blood Flow & Metabolism.

[0062] 40(5):939-953):

[0063] o Functional Ultrasound (fUS): A cutting-edge technique that uses high-frequency Doppler ultrasound to image rapid changes in cerebral blood volume with high spatial and temporal resolution. fUS is particularly useful for studying brain function and monitoring cerebral blood flow dynamics in response to different stimuli in real-time (Mace et al. 2011. Nat Methods 8, 662 664);

[0064] Single Photon Emission Computed Tomography (SPECT): Employs gamma cameras to detect emissions from radioactive isotopes like technetium-99m within the bloodstream, providing information on regional cerebral blood flow. SPECT is noted for its extensive use in assessing cerebral perfusion despite challenges in quantitative analysis (Yuko et al. 2012. Journal of Affective Disorders, 140 (3), 296-299):

[0065] • Positron Emission Tomography (PET): Uses positron-emitting isotopes such as oxygen- 15 or fluorine- 18, integrated into biologically active molecules.

[0066] PET tracks the distribution and concentration changes of these tracers, delivering both quantitative and qualitative data on cerebral perfusion and metabolism (Griffiths et al.2001. Vascular Medicine 6, 51-60}:

[0067] • Magnetic Resonance Imaging (MRI) Techniques: Use gadolinium -based contrast agents to assess cerebral blood flow. The technique calculates blood flow and volume from changes in the magnetic resonance signal as the contrast agents pass through the brain, offering high spatial resolution and the capability to examine brain function and structure simultaneously (Griffiths et al.2001. Vascular Medicine 6, 51-60}:

[0068] o Arterial Spin Labeling (ASL): A non-invasive MRI technique utilizing magnetically labeled arterial blood as an intrinsic tracer to measure CBF. It employs a radiofrequency pulse to invert the magnetization of arterial water protons before they enter brain tissue. ASL provides absolute quantification of CBF without the need for external contrast agents, suitable for patients unable to receive gadolinium-based agents (Chen 2011. Journal of Magnetic Resonance Imaging 33: 940 949; Wang et aL2019. BMC Pediatr. 19: 495)

[0069] o Dynamic Susceptibility Contrast (DSC) MRI: This technique involves the intravenous injection of gadolinium -based contrast agents. The passage of these agents through the cerebral vasculature causes changes in the magnetic resonance signal due to their magnetic properties. The technique is used to derive parameters such as cerebral blood volume (CBV) and CBF, useful in acute clinical settings such as stroke or tumor evaluation (Boxerman et al., 2020. Oncol. 22(9): 1262-1275):

[0070] o Dynamic Contrast-Enhanced MRI (DCE-MRI): This technique employs the intravenous administration of gadolinium-based contrast agents to study the dynamics of contrast perfusion through the tissues and blood vessels of the brain. By analyzing the rate and extent of the contrast agent's distribution and washout, DCE-MRI enables the quantification of parameters such as tissue permeability, blood volume, and vascular characteristics. This method is particularly useful in oncology for assessing tumor vascularity, guiding biopsy procedures, and monitoring therapeutic efficacy (Albano et al. 2022. Jpn J Radiol 40, 341 366; Tofts et al. 1999. Journal of Magnetic Resonance Imaging, 10(3): 223-232); o Blood Oxygenation Level-Dependent (BOLD) Functional MRI (fMRI): BOLD fMRI detects functional activity by observing changes in blood oxygenation and flow that occur in response to neural activity. It is predominantly used in research for mapping brain activity and understanding brain function, leveraging changes in blood deoxyhemoglobin concentration which affects the MR signal (Ogawa, et al. 1990. Proc. Natl Acad. Sci. USA 87, 9868-9872; Gore 2003. J. Clin. Investig. 112, 4-9).

[0071] • X-Ray Computed Tomography (CT) Perfusion: Involves the injection of iodinated contrast material and rapid sequential X-ray CT scanning. This technique tracks the passage of the contrast through the brain, with changes in tissue attenuation reflecting alterations in blood volume and flow (Griffiths etal.2001. Vascular Medicine 6, 51-60);

[0072] • Xenon-Enhanced CT (Xe-CT): Utilizes inhaled xenon gas that enters the bloodstream and brain, enhancing the CT image. Xenon's high solubility in blood and brain tissue allows it to serve as a freely diffusible tracer, providing quantitative measures of cerebral blood flow based on its concentration in different brain regions (Griffiths et al.2001. Vascular Medicine 6, 51-60);

[0073] • Near-Infrared Spectroscopy (NIRS): This non-invasive technique uses near-infrared light to measure cerebral blood flow and oxygenation within the brain's cortical layers. NIRS monitors changes in the absorption of near-infrared light caused by fluctuations in blood oxygen levels, providing insights into cerebral oxygenation and hemodynamics. This method is particularly valuable in studies of brain function during various cognitive tasks and for monitoring cerebral oxygenation in clinical settings (Strongman et al. 2002 NeuroImage, 17 (2): 719-731; Wolf et al. 2007. J Biomed Opt.

[0074] 12(6):062104).

[0075] In certain embodiments, the CBF level is measured using a method selected from the group consisting of computed tomography perfusion (CTP), dynamic susceptibility contrast magnetic resonance imaging (DSC-MRI), arterial spin labeling magnetic resonance imaging (ASL-MRI), single-photon emission computed tomography (SPECT), and transcranial Doppler ultrasound (TCD). In preferred embodiments, the CBF level is assessed using a method selected from the group consisting of CTP, DSC-MRI, and TCD. In more preferred embodiments, the CBF level is assessed using CTP or DSC-MRI. In the most preferred embodiments, the CBF level is assessed using CTP.

[0076] Certain embodiments of the invention relate to modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of cerebral blood flow said subject is suffering from or has a risk of suffering from a disease or medical condition selected from the group comprising a. neuroinflammatory and neurodegenerative diseases; particularly dementia selected from the group comprising mild cognitive impairment (MCI), Alzheimer’s disease, vascular dementia, mixed Alzheimer’s disease, and vascular dementia, Lewy body dementia, frontotemporal dementia, focal dementias (including progressive aphasia), subcortical dementias (including Parkinson’s disease), secondary causes of dementia syndrome (including intracranial lesions, multiple sclerosis, Huntington’s Disease, Amyloid Lateral Sclerosis, Traumatic Brain Injury, Creutzfeldt-Jakob Disease);

[0077] b. cardiovascular diseases or conditions, particularly selected from the group comprising atherosclerosis, hypertension, heart failure (including acute and acute decompensated heart failure), atrial fibrillation, cardiovascular ischemia, cerebral ischemic injury, cardiogenic shock, carotid artery stenosis, hypotension, hypertension, peripheral artery disease, coronary artery disease, stroke (including ischemic and haemorrhagic stroke and transient ischemic attack) and myocardial infarction;

[0078] c. metabolic disorders, particularly diabetes mellitus type 1, diabetes mellitus type 2, obesity, and metabolic syndrome;

[0079] d. infections caused by infectious organisms such as bacteria, viruses, fungi or parasites, particularly selected from the group comprising SIRS, sepsis, and septic shock;

[0080] e. respiratory disorders, particularly (chronic obstructive pulmonary disease, sleep apnea) f. psychiatric conditions (major depressive disorder, bipolar disorder).

[0081] Neuroinflammatory and neurodegenerative diseases prevented or treated according to embodiments of the present invention are selected from the group comprising mild cognitive impairment (MCI), Alzheimer’s disease (AD), vascular dementia, mixed Alzheimer’s disease, and vascular dementia, Lewy body dementia, frontotemporal dementia, focal dementias (including progressive aphasia), subcortical dementias (including Parkinson’s disease), secondary causes of dementia syndrome (including intracranial lesions, multiple sclerosis, Huntington’s Disease, Amyloid Lateral Sclerosis, Traumatic Brain Injury, Creutzfeldt-Jakob Disease)

[0082] AD is the most prevalent form of dementia. Key molecular mechanisms and histopathological hallmarks in the AD brain comprise a dynamic cascade of biochemical events including the pathological amyloidogenic cleavage of the amyloid precursor protein (APP), the generation of various beta-amyloid species including the amyloid-beta peptide (AP1-42), dimers, trimers, oligomers and subsequent amyloid aggregation and deposition in plaques, abnormal hyperphosphorylation and aggregation of tau protein, progressive intracellular neurofibrillary degeneration, changes within the innate immune system and inflammation and a breakdown of the blood-brain barrier.

[0083] MCI is a heterogeneous clinical condition with several underlying causes. However, the large proportion of MCI represents a transitional state between healthy aging and very mild AD (DeCarli 2003. Lancet Neurol. 2:15-21}. Accordingly, studies suggest that MCI subjects tend to progress to clinically probable AD at a rate of approximately 10%-l 5% per year (Markesbery 2010. J Alzheimers Dis. 19:221-228}. A breakdown of the blood-brain barrier in the hippocampus of MCI patients adds evidence to the hypothesis of BBB breakdown to precede neurodegeneration.

[0084] In preferred embodiments the patient group of Alzheimer's patients may be determined by risks factors such as the occurrence of MCI, the presence of the genetic risk factor ApoE4, as well as age. In a more preferred embodiment, the risk factor age of Alzheimer's patients is defined as an age of at least 60 years.

[0085] Dementia with Lewy bodies (DLB) is a type of dementia that worsens over time. DLB is associated with BBB dysfunction and microvascular lesions (Janelidze S etal. 2017. Neurobiol Aging. 51:104-112.}. Additional symptoms may include fluctuations in alertness, visual hallucinations, slowness of movement, trouble walking, and rigidity.

[0086] DLB is the most common cause of dementia after Alzheimer’s disease and vascular dementia. It typically begins after the age of 50. The underlying mechanism involves the formation of Lewy bodies in neurons, consisting of alpha-synuclein protein. A diagnosis may be suspected based on symptoms, with blood tests and medical imaging done to rule out other possible causes. At present no cure for DLB exists. For review see McKeith et al. 2017. Neurology 89: 88-100. Vascular dementia (VaD), also known as multi-infarct dementia (MID) and vascular cognitive impairment (VCI), is dementia caused by problems in the supply of blood to the brain, typically a series of minor strokes, leading to worsening cognitive decline that occurs step by step. The term refers to a syndrome consisting of a complex interaction of cerebrovascular disease and risk factors that lead to changes in the brain structures due to strokes and lesions and resulting in disruption of the blood-brain barrier and changes in cognition. The temporal relationship between a stroke and cognitive deficits is needed to make the diagnosis.

[0087] Frontotemporal dementia (FTD) is the clinical presentation of frontotemporal lobar degeneration, which is characterized by progressive neuronal loss predominantly involving the frontal or temporal lobes, and typical loss of over 70% of spindle neurons, while other neuron types remain intact. FTD is associated with BBB dysfunction and microvascular lesions (Janelidze S et al. 2017. Neurobiol Asins. 51: 104-112). an accounts for 20% of young-onset dementia cases. Signs and symptoms typically manifest in late adulthood, more commonly between the ages of 55 and 65, approximately equally affecting men and women. Common signs and symptoms include significant changes in social and personal behavior, apathy, blunting of emotions, and deficits in both expressive and receptive language. Currently, there is no cure for FTD, but there are treatments that help alleviate symptoms. For review see Bott et al. 2014. Neurodesener Dis Manas 4(6): 439 454.

[0088] The different dementia syndromes often show overlapping clinical features and related underlying pathology (known under the term “mixed Alzheimer’s disease”). In particular, Alzheimer’s dementia often co-occurs with vascular dementia. People with vascular dementia present with progressive cognitive impairment, acutely or sub-acutely as in mild cognitive impairment, frequently stepwise, after multiple cerebrovascular events (strokes). For review see Venkat et al. 2015. Exp Neurol 272: 97 108. Nevertheless, a pathology that is common for all forms of dementia is an impairment of the blood-brain barrier.

[0089] As used herein, “dementia " refers to substantially the clinical syndrome characterized by a cluster of symptoms and signs manifested by difficulties in memory, disturbances in language, psychological and psychiatric changes, and impairments in activities of daily living.

[0090] Therefore “dementia” may refer to Frontotemporal dementia, vascular dementia, Lewy body dementia, Alzheimer’s Dementia and Mild Cognitive Impairment and PAM according to the present invention may be used for treatment and / or prevention of cerebral hypoperfusion. In vascular cognitive impairment, CBF reductions follow a posterior-anterior gradient, impacting several regions but excluding the occipital lobe, indicating extensive white matter involvement that suggests subcortical circuit engagement. Multiple imaging modalities including SPECT, ASL-MRI, PET, and dynamic contrast-enhanced / dynamic susceptibility contrast MRI (DCE / DSC-MRI) are employed to quantify these changes (Park et al. 2014. Nucl Med Mol Imaging 48(4): 272-277; Yang et al. 2002. J Neurol Sci 203-204: 199-205; Sun et al. 2016. Front Aging Neurosci: 8: 211; Tohgi et al. 1998. Neuroradiology 40(3): 131-137). In the context of AD, multiple studies have demonstrated significant reduction of baseline CBF of ~ 10-20%, often preceding and predictive of the symptomatic onset of cognitive decline (Bracko et al., 2021. J Cereb Blood Flow Metab. 41(7): 1501 1516).

[0091] The progression of Alzheimer’s disease from its asymptomatic phase to dementia showcases varying degrees of CBF reduction. In the asymptomatic phase, middle-aged adults with a maternal history of Alzheimer's and ApoE4 carriers display regional blood flow differences, measured primarily using ASL-MRI (Okonkwo et al. 2014. Cereb Cortex 24(4): 978-988). As the disease progresses to MCI, reduced CBF is noted in regions including the occipital, angular gyrus, and posterior cingulate gyrus, among others, using ASL-MRI, GE-MRI, and other advanced techniques (Alexopoulos 2012. Eur Arch Psychiatry Clin Neurosci 262(1): 69 77; Dai et al. 2009. Radiology 250(3): 856-866; Johnson et al. 2005. Radiology 234(3): 851-859). The CBF reductions are also pronounced in the hippocampus and associated regions, areas crucial for memory and learning, thus directly impacting the cognitive deficits observed in AD. Moreover, a positive correlation between higher CBF and better cognitive function is observed (Swinford et al. 2022. medRxiv). The results from the in vivo rat model showed that chronic CBF reduction, induced by permanent bilateral occlusion of the common carotid arteries (PBOCCA), leads to cognitive dysfunction and neuroinflammatory responses. Morris water maze tests demonstrated impaired spatial learning and memory in the PBOCCA group compared to sham-operated controls. Histological analysis showed increased neuronal damage in the hippocampus of PBOCCA rats. Molecular studies revealed elevated gene expression of inflammatory markers such as NLRP3, caspase-1, ASC, and cathepsin B, indicating activation of the NLRP3 inflammasome pathway. Additionally, increased levels of cytokines IL-ip, IL- 18, IL-6, and Ap 1-42 were observed, suggesting an inflammatory response and AD-like pathology (Abedi et al. 2024, Neuroscience Research Notes: 7(2)). These findings highlight the role of chronic cerebral hypoperfusion in promoting neuroinflammation, neuronal damage, and cognitive impairment, relevant to the progression of Alzheimer's disease. In accordance with these findings, Korte et al. has shown, that the earliest detectable change in AD is a significant decrease in CBF, which occurs prior to cognitive symptoms and other pathological indicators like amyloid-beta deposition, which is primarily attributed to the constriction of capillaries, mediated by pericytes (Korte et al. 2020. Acta Neuropathol. 140(6):793-810).

[0092] Collectively, this evidence supports the hypothesis that cerebrovascular dysregulation might not just be a consequence of AD but a driving factor in its development, possibly mediating neurodegeneration via mechanisms of brain hypoperfusion and subsequent neuronal damage (Mokhber et al. 2021. The Neuroradiology Journal, Vol. 34(4): 300 307).

[0093] In the dementia stage, further reduction of CBF is seen, especially in the medial temporal and posterior cingulate gyrus, with limited compensatory increases in the anterior cingulate gyrus. A variety of imaging techniques including ASL-MRI and 7-tesla MRI are utilized (Hu et al.

[0094] 2010. Neurology 75(10): 881 888; Matsuda el al.2002. Eur J Nucl Med Mol Imaging 29(H): 1502-1505; Brundel et al. 2012. J Alzheimer s Dis 31(2): 259-263).

[0095] Further studies using two-dimensional phase-contrast magnetic resonance imaging (2D-PC MRI) have found that individuals with AD show a 20% lower CBF compared to age-matched non-demented controls, correlating strongly with lower cognitive performance scores and reduced hippocampal volumes (Leijenaar et al. 2017. Alzheimer s Dement (Amst). 9: 76-83). Focal dementias represent a subset of neurodegenerative disorders characterized by the progressive and selective impairment of specific cognitive functions, corresponding to localized brain region degeneration. Additionally, focal dementias initially affect discrete cognitive domains. Key types include Primary Progressive Aphasia (PPA), Frontotemporal Dementia (FTD), Corticobasal Degeneration (CBD), and Posterior Cortical Atrophy (PCA). PPA manifests through language deficits, with subtypes such as nonfluent / agrammatic, semantic, and logopenic variants. FTD primarily impacts behavior and executive functions, with behavioral variant FTD (bvFTD) and language variants overlapping with PPA. CBD presents with asymmetric motor symptoms and cognitive impairments, while PCA affects visuospatial and perceptual abilities. Pathophysiologically, focal dementias involve selective neuronal vulnerability to neurodegenerative processes, characterized by protein aggregation (e.g., tau, TDP-43, beta-amyloid), neuroinflammation, synaptic dysfunction, and genetic mutations (e.g., MAPT, GRN, C9orf72). Diagnostic approaches include neuroimaging (MRI, PET), neuropsychological testing, and biomarkers (CSF, blood). Subcortical dementias are a group of neurodegenerative disorders characterized by the predominant involvement of subcortical brain structures, including the basal ganglia, thalamus, and brainstem, leading to a distinct clinical syndrome. Unlike cortical dementias, which primarily affect higher-order cognitive functions, subcortical dementias are marked by a combination of cognitive, motor, and psychiatric symptoms. Key features include slowed cognitive processing, impaired executive functions, memory retrieval deficits, and motor abnormalities such as bradykinesia, rigidity, and gait disturbances. Prominent examples of subcortical dementias include Parkinson's disease dementia (PDD), Huntington's disease (HD), progressive supranuclear palsy (PSP), and vascular dementia with subcortical ischemic changes. The pathophysiology of subcortical dementias involves the degeneration of subcortical neurons, often associated with proteinopathies such as alpha-synuclein in PDD, huntingtin in HD, and tau in PSP. Additionally, vascular contributions to subcortical dementia involve chronic ischemia and small vessel disease, leading to white matter lesions and lacunar infarcts.

[0096] Neuroimaging techniques, including MRI and PET scans, are crucial for identifying characteristic subcortical atrophy and white matter changes. Neuropsychological assessments reveal patterns of cognitive impairment distinct from cortical dementias, emphasizing executive dysfunction and psychomotor slowing.

[0097] Parkinson’s disease (PD), the most common neurodegenerative disease of the elderly, is characterized by progressive loss of muscle control. PD is predominant at the 6th decade of life and men are 1.5 to 2 times more likely to contract the disease than women. Head trauma, illness, or exposure to environmental toxins is identified as a risk factor. This neurodegenerative disorder is characterized by tremor, rigidity, bradykinesia, and impairment in balance. PD also causes cognitive, psychiatric, autonomic, and sensory disturbances. The pathology of PD is characterized by the gradual and selective loss of dopaminergic neurons in the substantia nigra pars compacta.

[0098] Secondary causes of dementia encompass a range of conditions where cognitive decline arises as a consequence of underlying neurological or systemic disorders.

[0099] Intracranial lesions, including tumors, subdural hematomas, and abscesses, can disrupt normal brain function through mass effect, increased intracranial pressure, and direct neuronal damage, leading to cognitive deficits. Tumors, whether primary brain tumors such as gliomas and meningiomas or metastatic lesions, can cause cognitive decline by exerting pressure on adjacent brain structures, disrupting neural pathways, and causing edema. Subdural hematomas involve the accumulation of blood between the dura mater and the brain, leading to increased intracranial pressure and compression of brain tissue, resulting in cognitive and neurological deficits. Abscesses, which are infections leading to the formation of brain abscesses, can cause localized brain damage, inflammation, and subsequent cognitive impairment.

[0100] Multiple sclerosis (MS) is characterized by immune-mediated demyelination and axonal damage in the central nervous system. Lesions in white matter tracts disrupt communication between brain regions, leading to cognitive impairment.

[0101] Common cognitive deficits in MS include impaired memory, attention, information processing speed, and executive functions. These deficits correlate with lesion load and brain atrophy observed on MRI.

[0102] Huntington's disease (HD) is caused by a CAG trinucleotide repeat expansion in the HTT gene, leading to the production of mutant huntingtin protein. The disease primarily affects the basal ganglia, particularly the striatum, and extends to cortical regions as the disease progresses. Cognitive decline in HD includes difficulties with executive functions, memory retrieval, and visuospatial abilities, often accompanied by psychiatric symptoms such as depression and irritability.

[0103] Amyotrophic lateral sclerosis (ALS) involves the degeneration of upper and lower motor neurons, leading to progressive muscle weakness and atrophy. A subset of ALS patients develop frontotemporal dementia (ALS-FTD), characterized by changes in personality, behavior, and executive dysfunction. Pathologically, ALS-FTD is associated with TDP-43 proteinopathy. Traumatic brain injury (TBI) can result from blunt force trauma, acceleration-deceleration injuries, or penetrating injuries. Primary damage includes contusions and diffuse axonal injury, while secondary damage involves neuroinflammation, excitotoxicity, and hypoxia. Acute TBI can cause confusion, amnesia, and impaired attention. Chronic TBI, particularly repetitive injuries, can lead to chronic traumatic encephalopathy (CTE), characterized by progressive cognitive decline, mood disturbances, and behavioral changes.

[0104] Creutzfeldt-Jakob disease (CJD) is caused by the misfolding of prion proteins, leading to the accumulation of abnormal prions and widespread neuronal loss. CJD presents with rapidly progressive dementia, myoclonus, ataxia, and visual disturbances. The disease progresses rapidly, often leading to death within a year of onset. Diagnostic tools for CJD include MRI showing hyperintensities in the basal ganglia and cortical ribboning, EEG with periodic sharp wave complexes, and CSF biomarkers such as 14-3-3 protein. Neuroimaging, including MRI and PET scans, alongside cerebrospinal fluid analysis and genetic testing, are pivotal in diagnosing these conditions. Comprehensive neuropsychological assessments help delineate the specific cognitive deficits associated with each condition, aiding in differential diagnosis. Biomarkers such as 14-3-3 protein in CJD and genetic testing for HD provide supportive diagnostic information.

[0105] The pathophysiological mechanisms underlying these secondary causes of dementia involve proteinopathies, neuroinflammation, and genetic mutations.

[0106] Abnormal protein accumulations, such as alpha-synuclein in PDD, huntingtin in HD, and prions in CJD, play a central role in neuronal dysfunction and death. Chronic activation of glial cells and the release of inflammatory mediators contribute to neuronal damage in conditions like TBI and MS. Genetic mutations in genes such as HTT in HD and SOD1 in ALS are critical in the pathogenesis of these diseases.

[0107] Other neurodegenerative disorders such as Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FD) also demonstrate characteristic CBF patterns. Generalized CBF reduction across the cortex and subcortical areas is a feature of Amyotrophic Lateral Sclerosis, with specific reductions in the frontal and parietal lobes, assessed using ASL-MRI and other imaging modalities (Rule et al. 2010. Neurology 74(10): 821–827; Murphy et al. 2012. Amyotroph Lateral Scler 13(2): 202–209). Frontotemporal Dementia, on the other hand, shows reduced CBF in the frontal lobes but increased CBF in the medial parietal and posterior cingulate areas (Hu et al. 2010. Neurology 75(10): 881–888).

[0108] Huntington’s Disease and Lewy Body Dementia (LBD) exhibit reduced CBF in specific cortical regions such as the sensorimotor paracentral and occipital lobes, assessed mainly through ASL-MRI and SPECT respectively (Chen et al. 2012. Neuroimage 59(2):1043–1051; Hanyu et al.

[0109] 2006. J Neurol Sci 250(1-2): 97–10; Lobotesis et al. 2001. Neurology 56(5): 643–649). Multiple Sclerosis (MS) demonstrates reduced CBF in both white and gray matter. Moreover, MS is associated with increased blood-brain barrier permeability and impaired cerebrovascular reactivity, evaluated through techniques like DCE-MRI and ASL-MRI (Ingrisch et al. 2012. Invest Radiol 47(4): 252–258; Hojjat et al. 2016. AJNR Am J Neuroradiol 37(8): 1454–1461). Parkinson’s Disease is characterized by reduced CBF in a wide range of areas including the parietal, occipital, and frontal lobes, as well as microbleeds and impaired cerebrovascular reactivity, measured using T2-MRI, SWI-MRI, and ASL-MRI (Melzer et al. 2011. Brain 134(Pt 3): 845–855). Cardiovascular diseases prevented or treated by PAM according to embodiments of the present invention are, in particular, atherosclerosis, hypertension, heart failure (including acute and acute decompensated heart failure), atrial fibrillation, cardiovascular ischemia, cerebral ischemic injury, cardiogenic shock, carotid artery stenosis, hypotension, hypertension, peripheral artery disease, coronary artery disease, stroke (including ischemic and haemorrhagic stroke and transient ischemic attack) and myocardial infarction.

[0110] Cardiovascular diseases encompass a wide range of conditions affecting the heart and blood vessels.

[0111] Atherosclerosis is characterized by the accumulation of plaque within the arterial walls. Plaque, composed of lipids, cholesterol, calcium, and other substances, hardens and narrows the arteries over time, restricting blood flow. This can lead to serious complications such as myocardial infarction or stroke due to the reduced oxygen supply to vital organs.

[0112] Hypertension, or high blood pressure, is a condition where the force of the blood against the artery walls is consistently elevated. This increased pressure can damage blood vessels and lead to severe health problems, including heart disease, stroke, and renal failure. Hypertension often remains asymptomatic until significant damage has occurred, underscoring the importance of regular monitoring and management.

[0113] Heart Failure is a chronic condition where the heart's ability to pump blood is inadequate to meet the body's needs. It can be classified into:

[0114] o Acute Heart Failure: This involves a sudden onset of symptoms such as severe dyspnea, chest pain, and rapid or irregular heartbeat, requiring immediate medical attention.

[0115] o Acute Decompensated Heart Failure: This refers to a sudden exacerbation of chronic heart failure symptoms, often necessitating hospitalization. It is marked by severe respiratory distress, pulmonary edema, and a significant decline in cardiac function.

[0116] Atrial Fibrillation is an irregular and often rapid heart rate that can lead to thromboembolic events, including stroke. It occurs when the atria (upper chambers of the heart) beat irregularly and out of coordination with the ventricles (lower chambers). Symptoms may include palpitations, dyspnea, and fatigue.

[0117] Cardiovascular Ischemia involves reduced blood flow to the myocardium, typically due to partial or complete blockage of the coronary arteries. This condition can cause angina pectoris, myocardial infarction, or other serious cardiac conditions. The lack of oxygen-rich blood can damage the heart muscle and impair its function.

[0118] Cerebral Ischemic Injury results from insufficient cerebral blood flow, often leading to a stroke. This can occur when a blood vessel supplying the brain becomes occluded or narrowed, depriving brain cells of oxygen and nutrients. The resulting injury can cause loss of neurological function, disability, or death.

[0119] Cardiogenic Shock is a life-threatening condition where the heart suddenly cannot pump enough blood to meet the body's needs. Often precipitated by a severe myocardial infarction, it can also result from other cardiac conditions.

[0120] Symptoms include tachypnea, severe dyspnea, tachycardia, and loss of consciousness. Immediate medical intervention is crucial to prevent multi-organ failure and death.

[0121] Carotid Artery Stenosis is the narrowing of the carotid arteries, usually due to atherosclerosis. These arteries supply blood to the brain, and their narrowing can reduce cerebral blood flow, increasing the risk of ischemic stroke. Symptoms may include transient ischemic attacks (TIAs), which are temporary episodes of stroke-like symptoms.

[0122] Hypotension, or abnormally low blood pressure, can lead to dizziness, syncope, and inadequate perfusion of vital organs. While often less concerning than hypertension, chronic hypotension can indicate underlying health issues such as cardiac dysfunction, endocrine disorders, or severe infections.

[0123] Peripheral Artery Disease (PAD) is a circulatory problem where narrowed arteries reduce blood flow to the limbs, primarily the legs. This can cause intermittent claudication, numbness, and increased risk of infection in the affected limbs. PAD is often a sign of systemic atherosclerosis and can heighten the risk of myocardial infarction and stroke.

[0124] Coronary Artery Disease (CAD) results from the buildup of plaque in the coronary arteries, leading to their narrowing or occlusion. This reduces blood flow to the myocardium, causing angina pectoris, myocardial infarction, or heart failure. CAD is the most common type of heart disease and a leading cause of death worldwide.

[0125] Stroke is a medical emergency where the blood supply to part of the brain is interrupted or reduced, preventing brain tissue from receiving oxygen and nutrients. Strokes can be classified into: o Ischemic Stroke: Caused by an occlusion in an artery supplying blood to the brain, often due to a thrombus or embolus.

[0126] o Haemorrhagic Stroke: Caused by bleeding in or around the brain, often due to a ruptured blood vessel.

[0127] o Transient Ischemic Attack (TIA): Often called a mini-stroke, is a temporary period of symptoms similar to those of a stroke. TIAs do not cause permanent damage but are a warning sign of a future stroke.

[0128] Myocardial Infarction, commonly known as a heart attack, occurs when blood flow to a part of the myocardium is obstructed, leading to tissue ischemia and necrosis.

[0129] This is usually caused by an occlusion in one or more of the coronary arteries due to plaque rupture and thrombus formation. Symptoms include chest pain, dyspnea, nausea, and diaphoresis. Immediate medical treatment is crucial to restore blood flow and minimize myocardial damage.

[0130] Metabolic disorders prevented or treated by PAM according to embodiments of the present invention are, in particular diabetes mellitus type 1, diabetes mellitus type 2, obesity, and metabolic syndrome.

[0131] The metabolic syndrome was defined by the World Health Organization criteria (Alberti and Zimmet 1998. Diabet Med. 15:539–553) that require the presence of insulin resistance identified by one of the following: (1) type II diabetes; (2) impaired fasting glucose; (3) impaired glucose tolerance or (4) for those with normal fasting glucose levels (< 110 mg / dL), glucose uptake below the lowest quartile for background population under investigation under hyperinsulemic, euglycemic conditions, AND two of the following: (1) blood pressure: > 140 / 90 mmHg; (2) dyslipidemia: triglycerides (TG): > 1.695 mmol / L and high-density lipoprotein cholesterol (HDL-C) < 0.9 mmol / L (male), < 1.0 mmol / L (female); (3) central obesity: waist:hip ratio > 0.90 (male); > 0.85 (female), or body mass index > 30 kg / m2; (4) microalbuminuria: urinary albumin excretion ratio > 20 μg / min or albumin: creatinine ratio > 30 mg / g.

[0132] Diabetes Mellitus Type 1 is an autoimmune condition where the body's immune system attacks and destroys the insulin-producing beta cells in the pancreas, leading to little or no insulin production, which is essential for regulating blood glucose levels. Common symptoms include increased thirst, frequent urination, hunger, fatigue, and blurred vision. It is managed through lifelong insulin therapy, blood sugar monitoring, diet, and exercise. Diabetes Mellitus Type 2 is a chronic condition that affects the way the body processes blood sugar (glucose). It is characterized by insulin resistance, where the body's cells do not respond effectively to insulin, and eventually, the pancreas may not produce enough insulin. Symptoms include increased thirst, frequent urination, hunger, fatigue, and blurred vision. Management includes lifestyle changes (diet and exercise), oral medications, and sometimes insulin therapy. Obesity is a medical condition characterized by excessive body fat accumulation that presents a risk to health, typically measured by Body Mass Index (BMI), with a BMI of 30 or higher considered obese. It can lead to various health problems, including heart disease, diabetes, high blood pressure, and certain cancers. Management includes lifestyle changes such as diet and exercise, behavioral therapy, medications, and sometimes surgery.

[0133] Metabolic Syndrome is a cluster of conditions that occur together, increasing the risk of heart disease, stroke, and type 2 diabetes. These conditions include increased blood pressure, high blood sugar, excess body fat around the waist, and abnormal cholesterol or triglyceride levels. Often, there are no immediate symptoms, but a large waist circumference is a visible sign. Management focuses on addressing the individual risk factors through lifestyle changes (diet and exercise), medications, and regular monitoring.

[0134] Infections caused by infectious organisms such as bacteria, viruses, fungi or parasites prevented or treated by PAM according to embodiments of the present invention are selected from the group comprising SIRS, sepsis, and septic shock.

[0135] Systemic Inflammatory Response Syndrome (SIRS) is a clinical condition characterized by a systemic inflammatory response to various severe clinical insults, manifested by abnormalities in body temperature, heart rate, respiratory rate, and white blood cell count. SIRS can be triggered by both infectious and non-infectious causes.

[0136] When SIRS is due to an infection, it can progress to sepsis, which involves organ dysfunction. Non-infectious causes of SIRS include conditions such as pancreatitis, burns, trauma, and autoimmune disorders. The pathophysiology of SIRS involves the release of pro-inflammatory cytokines and other mediators that lead to widespread endothelial activation, increased vascular permeability, and the recruitment of immune cells to various tissues. This systemic inflammatory response can result in multiple organ dysfunction if not managed appropriately. Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. It occurs when the body’s response to an infection injures its own tissues and organs. Clinically, sepsis is characterized by the presence of infection and an acute change in the Sequential Organ Failure Assessment (SOFA) score of 2 or more points, reflecting the dysfunction of one or more organ systems. Common signs and symptoms include fever, tachycardia, tachypnea, and altered mental status. Sepsis can progress to septic shock, defined by persistent hypotension despite adequate fluid resuscitation, which can lead to multiple organ failure and increased mortality. Early recognition and prompt treatment with antibiotics and fluid resuscitation are critical for improving outcomes.

[0137] Septic shock is a severe and potentially fatal condition that occurs as a result of sepsis, characterized by persistent hypotension despite adequate fluid resuscitation. It is a state of profound circulatory, cellular, and metabolic abnormalities associated with a high risk of mortality.

[0138] Clinically, septic shock is defined by the following criteria:

[0139] 1. Sepsis: The presence of infection and an acute change in the Sequential Organ Failure Assessment (SOFA) score indicating organ dysfunction.

[0140] 2. Persistent hypotension: Blood pressure that remains low (usually defined as a systolic blood pressure of less than 90 mmHg, mean arterial pressure of less than 65 mmHg, or a decrease of 40 mmHg from baseline) despite appropriate fluid administration.

[0141] 3. Signs of organ dysfunction: This can include altered mental status, acute kidney injury, respiratory failure, and coagulopathy, among others.

[0142] Management typically involves aggressive fluid resuscitation, broad-spectrum antibiotics, and, if needed, vasopressor support to maintain adequate blood pressure and perfusion to vital organs. Early recognition and treatment are critical for improving survival rates.

[0143] Respiratory disorders prevented or treated by PAM according to embodiments of the present invention are selected from the group comprising Chronic Obstructive Pulmonary Disease (COPD) and Sleep Apnea.

[0144] Respiratory disorders encompass a broad range of medical conditions that affect the respiratory system, which includes the airways (nose, throat, bronchi), lungs, and the respiratory muscles involved in breathing. These disorders can lead to various symptoms, including difficulty breathing (dyspnea), cough, wheezing, and impaired gas exchange, which can ultimately result in decreased oxygenation of the blood and carbon dioxide retention. Examples include Chronic Obstructive Pulmonary Disease (COPD) and Sleep Apnea. Chronic Obstructive Pulmonary Disease (COPD) is a major respiratory disorder, primarily resulting from long-term exposure to harmful irritants, such as tobacco smoke. COPD includes chronic bronchitis, characterized by chronic airway inflammation and mucus production, and emphysema, which involves the destruction of alveoli, reducing gas exchange capacity. Common symptoms include chronic cough, sputum production, and shortness of breath. Diagnosis is typically confirmed through spirometry, which measures airflow limitations. Management focuses on symptom relief and slowing disease progression, emphasizing smoking cessation, bronchodilators, inhaled corticosteroids, and pulmonary rehabilitation. Sleep Apnea, particularly obstructive sleep apnea (OSA), involves repeated episodes of airway obstruction during sleep, leading to interrupted breathing and fragmented sleep. Patients often present with loud snoring, gasping during sleep, and excessive daytime sleepiness.

[0145] Diagnosis is made through polysomnography, which monitors various physiological parameters during sleep. Treatment options include lifestyle changes, continuous positive airway pressure (CPAP) therapy, oral appliances, and, in some cases, surgical intervention.

[0146] Psychiatric conditions prevented or treated by PAM according to embodiments of the present invention are selected from the group comprising major depressive disorder and bipolar disorder.

[0147] Psychiatric conditions encompass a diverse array of mental health disorders that significantly affect an individual's thoughts, emotions, behaviors, and overall functioning. These disorders can result from a complex interplay of genetic, biological, environmental, and psychological factors. Common examples include depression, anxiety disorders, schizophrenia, bipolar disorder, and personality disorders. Symptoms may manifest as mood disturbances, cognitive impairments, altered perceptions, and difficulties in interpersonal relationships. Diagnosis typically involves comprehensive assessments, including clinical interviews, standardized questionnaires, and, when necessary, neuropsychological testing. Treatment approaches are often multimodal, integrating psychotherapy, pharmacotherapy, and lifestyle modifications to address the specific needs of the patient.

[0148] Major Depressive Disorder (MDD) is a common mental health condition characterized by persistent feelings of sadness, hopelessness, and a lack of interest or pleasure in nearly all activities. To be diagnosed, individuals typically must experience at least five of the following symptoms for a duration of at least two weeks: depressed mood, loss of interest or pleasure, significant weight change, insomnia or hypersomnia, psychomotor agitation or retardation, fatigue or loss of energy, feelings of worthlessness or excessive guilt, difficulty concentrating, and recurrent thoughts of death or suicidal ideation. MDD can lead to significant impairment in social, occupational, and other areas of functioning. The exact etiology is multifactorial, involving genetic, biochemical, environmental, and psychological factors. Treatment often includes a combination of psychotherapy, pharmacotherapy (such as antidepressants), and lifestyle changes.

[0149] Bipolar Disorder is a mental health condition characterized by significant mood swings that include emotional highs (mania or hypomania) and lows (depression). During manic or hypomanic episodes, individuals may experience increased energy, heightened mood, reduced need for sleep, impulsivity, and racing thoughts. Conversely, depressive episodes involve persistent feelings of sadness, hopelessness, and loss of interest in activities, similar to those seen in Major Depressive Disorder.

[0150] The exact cause of bipolar disorder is complex and involves a combination of genetic, biological, and environmental factors. Diagnosis typically requires a thorough assessment of the individual's mood patterns and functional impairments over time. Treatment often involves mood stabilizers, antipsychotic medications, psychotherapy, and lifestyle adjustments to manage symptoms and prevent relapse. Effective management of bipolar disorder aims to stabilize mood and improve overall functioning while minimizing the impact of episodes on daily life.

[0151] In particular embodiments of the invention, said subject is suffering from or has a risk of suffering from a disease or medical condition selected from the group consisting of MCI, Alzheimer’s disease, mixed Alzheimer’s disease, and vascular dementia.

[0152] In further particular embodiments of the invention, said subject is suffering from or has a risk of suffering from a disease or medical condition selected from the from the group consisting of cerebral ischemic injury, stroke (including ischemic and haemorrhagic stroke and transient ischemic attack).

[0153] In further particular embodiments of the invention, said subject is suffering from or has a risk of suffering from a disease or medical condition selected from the from the group consisting of ischemic stroke, haemorrhagic stroke and transient ischemic attack (TIA).

[0154] In further particular embodiments of the invention, said subject is suffering from or has a risk of suffering from a disease or medical condition selected from the from the group consisting of sepsis and septic shock. As used herein, “PAM” includes all isoforms of PAM, in particular PAM-1, PAM-2, PAM-3, PAM-4, PAM-5 and PAM-6 (SEQ ID No. 1 to SEQ ID No. 6).

[0155] It is to be understood by the skilled artisan, that the PAM isoform sequences (SEQ ID No. 30 to 35) as represented in the sequence list, contain an N-terminal signal sequence (amino acid 1-20) and pro-region (amino acid 21 to 30). This N-terminal signal sequence and pro-region is cleaved off prior to secretion of the protein. Therefore, in preferred embodiments the PAM isoform sequences (SEQ ID No. 1 to 6) and / or fragments thereof do not contain the N-terminal signal sequence. Moreover, the person skilled in the art understands that all isoforms of PAM catalyse the conversion of peptides with C-terminal glycine amino acid into alpha-ami dated peptides leading to a biological activation of these hormones. These isoforms only exhibit differences in tissue distribution, enzymatic activity, and regulatory properties.

[0156] Certain embodiments of the invention relate to the modified or unmodified fragments of PAM according to SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 9 for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of cerebral blood flow (CBF).

[0157] Certain embodiments of the invention relate to the modified or unmodified fragments of PAM according to SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 9 for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of cerebral blood flow wherein said subject is suffering from or has a risk of suffering from a disease or medical condition selected from the group consisting of mild cognitive impairment (MCI), Alzheimer’s disease, mixed Alzheimer’s disease, and vascular dementia.

[0158] Certain embodiments of the invention relate to the modified or unmodified fragments of PAM according to SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 9 for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of cerebral blood flow wherein said subject is suffering from or has a risk of suffering from a disease or medical condition selected from the group consisting of cerebral ischemic injury, stroke (including ischemic and haemorrhagic stroke and transient ischemic attack).

[0159] Certain embodiments of the invention relate to the modified or unmodified fragments of PAM according to SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 9 for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of cerebral blood flow wherein said subject is suffering from or has a risk of suffering from a disease or medical condition selected from the group consisting of ischemic stroke, haemorrhagic stroke and transient ischemic attack (TIA).

[0160] Certain embodiments of the invention relate to the modified or unmodified fragments of PAM according to SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 9 for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of cerebral blood flow wherein said subject is suffering from or has a risk of suffering from a disease or medical condition selected from the group consisting of sepsis and septic shock. In certain specific embodiments of the invention, the modified or unmodified PAM or fragments thereof are isolated from a tissue or organ or are recombinantly produced as wild-type PAM, modified PAM, or chimeric PAM. In further embodiments of the invention, the modified or unmodified PAM or fragments thereof are recombinantly expressed using a recombinant system selected from the group comprising:

[0161] • Mammalian cell lines, including but not limited to CHO-K1, CHO-S, and CHO-DG4;

[0162] • Yeast systems, including but not limited to Pichia pastoris strains X-33, GS115, and KM71;

[0163] • Bacterial systems, including but not limited to Escherichia coli strains BL21(DE3), Rosetta(DE3), and Shuffle;

[0164] • Insect cell systems, including but not limited to baculovirus expression systems using cell lines such as Sf9, Sf21, and High Five;

[0165] • Plant-based systems, including but not limited to Nicotiana benthamiana, BY-2, and rice cell cultures;

[0166] • Cell-free expression systems, including but not limited to wheat germ extract, rabbit reticulocyte lysate, and E. coli S30 extract.

[0167] In specific embodiments of the invention, the modified or unmodified PAM or fragments thereof are recombinantly expressed in CHO or E. coli cells.

[0168] In certain specific embodiments of the invention, the modified or unmodified PAM or fragments thereof are purified from an organ or tissue selected from the group comprising heart, brain (specifically hypothalamus, hippocampus, or pituitary glands), blood, plasma, adrenal glands, thyroid glands, epithelial tissue (specifically endothelium and endometrium), pancreas, and kidney. In certain specific embodiments of the invention, the modified or unmodified PAM or fragments thereof are purified from blood, plasma, or brain.

[0169] In certain embodiments said PAM or fragments thereof are isolated from a cell or tissue (including bodily fluids, e.g. blood) / organ or are recombinantly produced or are chimeric PAM or fragments thereof. Said cell or tissue / organ may be selected from mammalians, bacteria, yeast, fungi etc.

[0170] PAM for use according to the present invention can be a commercial PAM enzyme, or any formulation comprising the PAM enzyme and any means capable of producing a functional PAM enzyme in the context of the current invention, such as DNA or RNA nucleic acids encoding a PAM protein. The nucleic acid encoding PAM may be embedded in suitable vectors such as plasmids, phagemids, phages, (retro)viruses, transposons, gene therapy vectors and other vectors capable of inducing or conferring production of PAM. Also native or recombinant microorganisms, such as bacteria, fungi, protozoa and yeast may be applied as a source of PAM in the context of the current disclosure.

[0171] In some embodiments, the mammalian PAM is a human, a porcine or a bovine PAM.

[0172] In certain embodiments said PAM is a protein comprising an amino acid sequence selected from the group comprising SEQ ID Nos. 1 to 10, or an amino acid sequence having at least 85%, particularly at least 90%, more particularly at least 95%, even more particularly at least 99% sequence identity therewith.

[0173] In certain embodiments, the PAM is a functional fragment (i.e., PHM (SEQ ID No. 7) and PAL (SEQ ID No. 8), PAM conserving at least about 85%, at least about 90%, at least about 95%, or at least about 99% of the PAM activity of the corresponding functional fragment of PAM. In some embodiments, the PAM is a variant or a derivative of PAM disclosed herein.

[0174] More particularly, a fragment of PAM is a functional fragment having an amino acid sequence consisting of between 80 and 100%, more particularly between 85 and 100%, more particularly between 90 and 100%, even more particularly between 95 and 100%, yet even more particularly between 98 and 100% of SEQ ID Nos. 1 to 10; where necessary, the resulting amino acid numbers of said fragments are rounded down. As an example, between 98 and 100% of a 970 amino acid sequence corresponds to 950 (950.6 rounded down) to 969 amino acids (969.999 rounded down). The percentage of identity of an amino acid or nucleic acid sequence, or the term “% sequence identity”, is defined herein as the percentage of residues in a candidate amino acid or nucleic acid sequence that is identical with the residues in a reference sequence after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent identity.

[0175] In preferred embodiments, the calculation of said at least percentage of sequence identity is carried out without introducing gaps. Methods and computer programs for the alignment are well known in the art, for example “Align 2” or the BLAST service of the National Center for Biotechnology Information (NCBI).

[0176] In certain embodiments of the invention said PAM or fragment thereof is enzymatically active. As used herein, “enzymatically active” refers to a protein or modified form of such protein or a fragment of such protein or modified form thereof, thereof that retains or essentially retains the proteins enzymatic activity. In the case of PAM, the enzymatic activity of refers in particular to the alpha-amidating activity of PAM.

[0177] The term “unmodified PAM” refers to all isoforms and single subunits of PAM, in particular those based on the SEQ ID Nos. 1 to 10, and which have not undergone any deliberate or artificial modifications, including chemical conjugation, alteration of the amino acid sequence, including amino acid exchange, deletion or insertion, fusion with other proteins, or other such methods. While there can be certain variations depending on its source organism, conditions of expression and / or purification, as well as certain naturally occurring mutants and variants known in the field, in particular unmodified PAM has an amino acid sequence of naturally occurring PAM and more particularly at least essentially retains its natural three-dimensional structure, function, and properties.

[0178] The term “modified PAM” refers to all isoforms and single subunits of PAM, in particular those based on the SEQ ID Nos. 1 to 10, which are modified in accordance with the present invention, in particular as detailed herein, more particularly by amino acid manipulation, by fusion to other proteins, such as albumin, e.g. serum albumin or recombinant serum albumin, by non-covalent binding to serum albumin via a conjugated fatty acid chain to PAM, via fusion with IgG Fc regions or transferrin, via post-translational modification attaching natural or synthetic polymers, wherein the natural or synthetic polymer to be used is particularly HAP, more particularly ELP, more particularly PAS, more particularly PSA, more particularly GLK, more particularly XTEN and even more particularly PEG.

[0179] Modifications of PAM may be selected from the group comprising: • Amino acid manipulation, also referred to as site directed mutagenesis, including the insertion, deletion or alteration of one or more amino acids within the polypeptide amino acid sequence reducing immunogenicity and proteolytic instability in vivo. Thereby alterations of one or more amino acids may lead to enhanced protease resistance in vivo.

[0180] • Conjugation and / or bioconjugation of polypeptides of interest with serum proteins, such as albumins or immunoglobulins or parts of immunoglobulins to produce fusion proteins: • Fusion of polypeptides with Albumins, e.g. serum Albumin or recombinant serum Albumin.

[0181] • Non covalent binding to serum Albumin due to a conjugated fatty acid chain to a polypeptide of interest. The binding to albumin therefore happens in vivo after application of the fatty acid conjugated substance and is mediated by the fatty acid chain.

[0182] • Fusion of polypeptides with IgG Fc regions or Transferrin.

[0183] • Post-translational modifications attaching natural or synthetic polymers to the polypeptide of interest. Example for such polymers, but not limited to, are

[0184] • PEG, either single-stranded or branched PEG, having varying molecular weights covalently fused to polypeptides of interest.

[0185] • XTEN, an unstructured polypeptide, covalently fused to polypeptides of interest. XTEN is a 864 single amino-acid sequence composed of amino-acids Ala, Glu, Gly, Pro, Ser and Thr in a randomized manner. Half-life of an XTEN-fusion protein may be tailored by shortening of the XTEN sequence.

[0186] • PAS, which is a peptide polymer consisting of amino acids proline, alanine and serine wit 100-200 PAS repeats forming the polymer.

[0187] • ELP (Elastin-like polypeptides) consisting of Valin-Prolin-Glycine-x-Glycine repeats, naturally found in elastin, wherein x relates to any amino-acid except for Proline. ELP can be covalently attached to a polypeptide of interest.

[0188] HAP, which is a repeated sequence of glycine rich (Gly4Ser)n polypeptide, wherein n is in-between of 100-200 and is covalently attached to the protein of interest. • GLK, which is a gelatin-like fusion protein. Thereby GLK is a (Gly-X-Y)n structure, wherein X and Y are any amino-acids except for Cysteine, with n = 60 to 1500. GLK can be covalently attached to a polypeptide of interest.

[0189] • Carbohydrates and polysaccharides. Carbohydrates, either branched or linear can be attached to the polypeptide of interest e.g. through in vivo N-Glycosylation. Conjugation with Dextrans, Hydroxy ethyls (HES), Heparosan (HEP), Hyaluronic acid (HA) represents the attachment of polysaccharides.

[0190] • PSA (Polysialic acid), wherein PSA polymers are covalently attached to the polypeptide of interest.

[0191] In certain embodiments of the invention said PAM or fragments thereof are modified by a. attaching one or more natural or synthetic polymers units, wherein said polymers are selected from the group comprising

[0192] i. PEG, particularly having an average molecular weight in the range of 0.2-100 kDa, particularly 1-90 kDa, more particularly 2-80 kDa, more particularly 3-70 kDa, more particularly 4-60 kDa and even more particularly 5 to 50kDa;

[0193] ii. XTEN;

[0194] iii. a peptide polymer consisting of repeats of PAS (the amino acids proline, alanine and serine, particularly comprising 100-200, particularly 120-180, more particularly 130- 170, even more particularly about 150 PAS repeats;

[0195] iv. elastin-like polypeptides consisting of Valin-Prolin-Glycine-x-Glycine repeats, wherein x is any amino acid other than Proline, particularly consisting of from 150, 200, 250, 300, 400, or 500 to 550, 600, 750, 850, 950 or 1,000 amino acids; v. HAP polypeptide, having the sequence (Gly4Ser)n, wherein n is 100-200;

[0196] vi. gelatin-like fusion protein, having the sequence (Gly-X-Y)n, wherein X and Y individually are any amino acid except for Cysteine, and wherein n = 60 to 1500; vii. polysaccharides, in particular those chosen from the group comprising:

[0197] 1. dextrans;

[0198] 2. hydroxyethyls; 3. heparosan;

[0199] 4. hyaluronic acid;

[0200] viii. poly sialic acid.

[0201] b. conjugation with a serum protein, such as albumin or an immunoglobulin or parts of an immunoglobulin;

[0202] c. site-directed mutagenesis by insertion, deletion and / or exchange of one or more amino acids within the amino acid sequence of PAM or fragments thereof.

[0203] In more specific embodiments of the invention said PAM or fragments thereof are modified by attaching one or more natural or synthetic polymers units, wherein said polymers are selected from the group consisting of

[0204] i. PEG, particularly having an average molecular weight in the range of 0.2-100 kDa, particularly 1-90 kDa, more particularly 2-80 kDa, more particularly 3-70 kDa, more particularly 4-60 kDa and even more particularly 5 to 50 kDa; or

[0205] ii. XTEN; or

[0206] iii. HAP polypeptide, having the sequence (Gly4Ser)n, wherein n is 100-200.

[0207] In certain specific embodiments of the invention said PAM or fragments thereof are modified by attaching one or more polymer units, wherein said polymer is PEG.

[0208] In certain specific embodiments of the invention said polymer is PEG having an average molecular weight of 5 to 50 kDa.

[0209] In other more preferred embodiments, modification of PAM is modification with PEG (PEGylation), preferably PEG with an average molecular weight of 5-10 kDa (PEG 5000 to PEG 10.000) or, more preferably with an average molecular weight of about 5 kDa (PEG 5000). In further embodiments, modification of PAM with PEG (PEGylation) shall mean the attachment of PEG 5000 or 10000 as detailed herein to serine or lysine side chains of PAM, more preferably the attachment of PEG 10000 as detailed herein to lysine side chains of PAM, or more preferably the attachment of PEG 5000 as detailed herein to lysine or serine, even more preferably to serine side chains of PAM. In other more preferred embodiments, modification of PAM is modification with XTEN as defined herein, preferably with XTEN having a sequence of SEQ ID No: 36 more preferably to lysine or serine side chains of PAM, more preferably to lysine side chains of PAM, or alternatively via a Cys amino acid C-terminally added to PAM.

[0210] In certain embodiments, modification of PAM with PEG (PEGylation) shall mean the attachment of PEG as detailed herein to lysine side chains, in particular in alkaline aqueous solution, e.g. in a buffered solution, particularly at a pH from 7.75 to 9.25, more particularly from 8.25 to 8.75, more particularly about 8.5; in particular with a molar excess of PEG polymer vs. PAM of 70- to 140-fold, more particularly 80- to 130-fold, more particularly 90- to 120-fold; in particular embodiments with a method analogous to the method described in Example 5, which can be adapted to other PAM types and / or other PEG polymers by routine methods. The PEGylation rate and molecular weight of the resulting PEGylated PAM can be analyzed by various well-known methods, such as gel filtration or SDS-PAGE.

[0211] Modifications of PAM may also include modifications, which allow PAM to act as a prodrug. The term "prodrug" denotes a form or derivative of a compound which is metabolized in vivo, e.g., by biological fluids or enzymes by a subject after administration, into a pharmacologically active form of the compound in order to produce the desired pharmacological effect.

[0212] Prodrugs can thus be viewed as drugs containing specialized non-toxic protective groups used in a transient manner to alter or to eliminate undesirable properties in the parent molecule. The group of the afore mentioned prodrugs may comprise Carrier-linked prodrugs (Carrier prodrugs), Cascade prodrugs and PEG-based carrier prodrugs. A carrier-linked prodrug may be a prodrug that contains a temporary linkage of a given active substance with a transient carrier group that produces improved physicochemical or pharmacokinetic properties and that can be easily removed in vivo, usually by a hydrolytic cleavage, wherein a cascade prodrug is a prodrug for which the cleavage of the carrier group becomes effective only after unmasking an activating group and wherein several examples of PEG-based carrier prodrugs exist, most of them with the need for enzymatic activation of the linker between the active drug and the carrier, mostly initiated by enzymatic hydrolysis. Since esters are cleaved very readily and unpredictably in vivo, direct ester linkers for carrier pro drug have limitations to their usability (J. Rautio etal.. Nature Reviews Drug discovery, 2008, 7 255-270).

[0213] Modified PAM may refer to PAM (SEQ ID No.: 1-10) modified via amino acid manipulations, preferably via fusion to Albumins, e.g. serum Albumin or recombinant serum Albumin, more preferably via non covalent binding to serum Albumin due to a conjugated fatty acid chain to PAM, more preferably via fusion with IgG Fc regions or Transferrin, most preferably via post-translational modifications attaching natural or synthetic polymers, whereas the natural or synthetic polymer to be used is HAP, preferably ELP, more preferably PAS, more preferably PSA, more preferably GLK, more preferably XTEN and most preferably PEG.

[0214] PEG-PAM may refer to PAM (SEQ ID No.: 1-10) modified with 1+n molecules of polyethylene glycol (PEG), whereas n is an integer in the range of 0 to 100 and one PEG molecule has a molecular weight in the range of 1-100 kDa and is either a linear molecule or a branched molecule with b+1 branches, whereas b is an integer in the range of 0 to 20.

[0215] PAM modified via site directed mutagenesis may refer to PAM (SEQ ID No.: 1-10) with an insertion of n+1 additional amino-acids and / or n+1 deletions of amino-acids and / or n+1 exchanged amino-acids in the given amino-acid sequence (SEQ ID No.: 1-10), wherein n is an integer in the range of 0-100.

[0216] PAM modified via fusion to native serum Albumin or recombinant human serum Albumin may refer to PAM (SEQ ID No.: 1-10) fused to native human serum Albumin or recombinant human serum Albumin at the N-terminus or at the C-terminus of PAM according to (SEQ ID No.: 1-10).

[0217] PAM modified via fusion to IgG Fc regions may refer to PAM (SEQ ID No.: 1-10) fused to human IgGl Fc Region at the N-terminus or at the C-terminus of PAM according to (SEQ ID No.: 1-10).

[0218] PAM modified via XTEN may refer to PAM (SEQ ID No.: 1-10) fused to a XTEN moiety at the N-terminus or at the C-terminus of PAM according to (SEQ ID No.: 1-10) and / or to any surface exposed amino-acid of PAM according to (SEQ ID No.: 1-10).

[0219] Fusion may refer to a covalent or non-covalent linkage of two proteins or polypeptides, a protein and a polypeptide, a synthetic polymer and a polypeptide and / or a synthetic polymer and a protein to each other. In preferred embodiments fusion may refer to covalent linkage.

[0220] A person skilled in the art knows how to create an expression vector for expression of N-terminal or C-terminal fusion proteins. The person skilled in the art knows that fusion of two proteins can be achieved by usage of state-of-the-art techniques to result in a covalent or non-covalent linkage of two proteins, a protein and a polypeptide or a protein and / or a polypeptide and a synthetic polymer. The person skilled in the art knows how to perform site directed mutagenesis. In terms of this invention, the biological activity of the modified PAM shall remain present to an extent of at least 1 % (in particular 1-300%) when directly compared to the non-modified active moiety, preferably of at least 5% (in particular 5-300%), more preferably of at least 15% (in particular 15-300%), more preferably of at least 30% (in particular 30-300%), more preferably of at least 50% (in particular -300%) more preferably of at least 70% (in particular 70-300%), more preferably of at least 90% (in particular 90-300%), more preferably of at least 150% (in particular 150-300%), more preferably of at least 200% (in particular 200-300%) and most preferably of at least 250% (in particular 250 -300%). In terms of modified PAM, biological activity shall mean the capability of modified PAM to perform C-terminal amidation and may be tested using PAM activity assays with synthetic, labelled tripeptides, such as Tyr-Val-Gly as substrate. Preferably the activity is determined as described in Example 4 using wildtype ADM-Gly as substrate.

[0221] In certain embodiments said PAM or fragment thereof is administered in combination with ascorbate and / or copper and / or peptide with C-terminal Glycine amino acid or fragments thereof.

[0222] In certain embodiments of the invention said peptide with C-terminal Glycine amino acid is selected from the group of comprising adrenomedullin (ADM), adrenomedullin-2, intermedinshort, pro-adrenomedullin N-20 terminal peptide (PAMP), amylin, gastrin-releasing peptide, neuromedin C, neuromedin B, neuromedin S, neuromedin U, calcitonin, calcitonin gene-related peptide (CGRP) 1 and 2, islet amyloid polypeptide, chromogranin A, insulin, pancreastatin, prolactin-releasing peptide (PrRP), cholecystokinin, big gastrin, gastrin, glucagon-like peptide 1 (GLP-1), pituitary adenylate cyclase-activating polypeptide (PACAP), secretin, somatoliberin, peptide histidine methionine (PHM), vasoactive intestinal peptide (VIP), gonadoliberin, kisspeptin, MIF-1, metastin, neuropeptide K, neuropeptide gamma, neuropeptide Y, substance P, neurokinin A, neurokinin B, peptide YY, pancreatic hormone, deltorphin I, orexin A and B, melanotropin alpha (alpha-MSH), melanotropin gamma, thyrotropin-releasing hormone (TRH), oxytocin, vasopressin or fragments thereof.

[0223] Certain embodiments of the invention relate to modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF, wherein PAM is combined with Vitamin C.

[0224] Certain embodiments of the invention relate to modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF, wherein PAM is combined with a peptide with C-terminal Glycine amino acid, in particular ADM, VIP, PACAP, GLP-1 and neuropeptide Y or fragments thereof.

[0225] Certain embodiments of the invention relate to modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF, wherein said fragments of ADM-Gly (SEQ ID No. 14) are selected from the group of ADM-Gly 2-53 (SEQ ID No. 17), ADM-Gly 8-53 (SEQ ID No. 18), ADM-Gly 22-53 (SEQ ID No. 19), ADM-Gly 27-53 (SEQ ID No. 20) and ADM-Gly 33-53 (SEQ ID No. 21).

[0226] Certain embodiments of the invention relate to PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF, wherein PAM is to be used in combination with ADM-Gly or fragments thereof and in addition is to be used with Vitamin C.

[0227] Certain embodiments of the invention relate to PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF, wherein PAM is to be used in combination with ADM-Gly or fragments thereof and in addition is to be used with Vitamin C, wherein administered ADM-Gly or fragments thereof is amidated by administered PAM in vivo. Moreover, said combination leads to a sustained elevation of bio- ADM.

[0228] The effect of said combination is the prolonged elevation of resulting bio-ADM in circulation in comparison to the short bioavailability of wildtype-bio-ADM when administered alone. Other than in the state of the art, an elevation of active bio-ADM levels in circulation is reached without modifications of ADM-Gly or fragments thereof in said combination. Therefore, said combinations according to the present invention are suitable for treatment and / or prevention of cerebral hypoperfusion.

[0229] In other certain embodiments of the invention said peptide with C-terminal glycine amino acid is selected from the group consisting of ADM, VIP, PACAP, GLP-1 and neuropeptide Y or fragments thereof.

[0230] In the context of the present invention, prevention of cerebral hypoperfusion includes prevention of a reduction of cerebral blood flow. For instance, a patient having a cerebral blood flow that is below the normal range (e.g. 50 mL / lOOg (of brain tissue) per min) but to a limited extent, e.g. less than about 5 % or less than about 10 % below the normal range, may have a risk for further reduction in CBF. The administration of modified or unmodified PAM or fragments thereof may then prevent further reduction in CBF.

[0231] In particular embodiments of the present invention, said PAM is administered orally, epicutaneously, subcutaneously, intradermally, sublingually, intramuscularly, intraarterially, intravenously, via the central nervous system (CNS, intracerebrally, intracerebroventricularly, intrathecally) or via intraperitoneal administration, particularly epicutaneously, subcutaneously, intradermally, intramuscularly, or intraperitoneally, more particularly subcutaneously, intramuscularly, or intraperitoneally; “formulated to be administered (e.g. intramuscularly)” likewise reads on “formulated for (e.g. intramuscular) administration”.

[0232] In preferred embodiments of the present invention, said PAM is administered epicutaneously, subcutaneously, intradermally, intramuscularly, intraarterially, intravenously, via the central nervous system (CNS, intracerebrally, intracerebroventricularly, intrathecally) or via intraperitoneal administration, particularly epicutaneously, subcutaneously, intradermally, intramuscularly, or intraperitoneally, more particularly subcutaneously, intramuscularly, or intraperitoneally; “formulated to be administered (e.g. intramuscularly)” likewise reads on “formulated for (e.g. intramuscular) administration.”

[0233] In the most preferred embodiments of the present invention, said PAM is administered subcutaneously, intradermally, intramuscularly, intraarterially or intravenously, or via intraperitoneal administration.

[0234] In specific embodiments of the invention said PAM is administered subcutaneously or intravascular (intraarterially, intravenously).

[0235] Pharmaceutical formulation

[0236] Further subject matter of the present invention is a pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF.

[0237] In certain embodiments said subject that has a reduction in cerebral blood-flow.

[0238] In certain embodiments said PAM or fragments thereof are isolated from a cell or tissue (including bodily fluids, e.g. blood) / organ or are recombinantly produced or are chimeric PAM or fragments thereof. Said cell or tissue / organ may be selected from mammalians, bacteria, yeast, fungi etc. In certain embodiments said PAM or a fragment thereof is a protein comprising an amino acid sequence selected from the group comprising SEQ ID Nos 1 to 10, or an amino acid sequence having at least 85%, particularly at least 90%, more particularly at least 95%, even more particularly at least 99% sequence identity therewith.

[0239] In certain embodiments said PAM or fragment thereof is enzymatically active.

[0240] In certain embodiments said PAM or a fragment thereof is modified by

[0241] a. attaching one or more natural or synthetic polymers units, wherein said polymers are selected from the group comprising

[0242] iv. PEG, particularly having an average molecular weight in the range of 0.2-100 kDa, particularly 1-90 kDa, more particularly 2-80 kDa, more particularly 3-70 kDa, more particularly 4-60 kDa and even more particularly 5 to 50kDa; v. XTEN;

[0243] vi. a peptide polymer consisting of repeats of PAS (the amino acids proline, alanine and serine, particularly comprising 100-200, particularly 120-180, more particularly 130-170, even more particularly about 150 PAS repeats;

[0244] vii. elastin-like polypeptides consisting of Valin-Prolin-Glycine-x-Glycine repeats, wherein x is any amino acid other than Proline, particularly consisting of from 150, 200, 250, 300, 400, or 500 to 550, 600, 750, 850, 950 or 1,000 amino acids; viii. HAP polypeptide, having the sequence (Gly4Ser)n, wherein n is 100-200; ix. gelatin-like fusion protein, having the sequence (Gly-X-Y)n, wherein X and Y individually are any amino acid except for Cysteine, and wherein n = 60 to 1500; x. polysaccharides, in particular those chosen from the group comprising: 1. dextrans;

[0245] 2. hydroxyethyls;

[0246] 3. heparosan;

[0247] 4. hyaluronic acid;

[0248] xi. polysialic acid.

[0249] b. conjugation with a serum protein, such as albumin or an immunoglobulin or parts of an immunoglobulin;

[0250] c. site-directed mutagenesis by insertion, deletion and / or exchange of one or more amino acids within the amino acid sequence of PAM. In certain embodiments, the pharmaceutical formulation according to the invention comprises one or more items from the group comprising packaging, instructions to the subject and / or physician treating the subject and a leaflet. Such items may in particular comprise instructions regarding the administration of the pharmaceutical formulation to a subject, such as relating to the dosage, medical indications, administration route.

[0251] The embodiments of the present invention relating to the modified or unmodified PAM or pharmaceutical formulation relating to the modified or unmodified PAM for use in treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF analogously relate to the following: Certain embodiments of the present invention relate to the use of modified or unmodified PAM for the manufacture of a medicament for the treatment of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF. Certain embodiments of the present invention relate to the use of modified or unmodified PAM for the treatment of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF. Certain embodiments of the present invention relate to modified or unmodified PAM for the treatment of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF. In particular embodiments of the present invention, said pharmaceutical formulation is formulated to be administered or is administered orally, epicutaneously, subcutaneously, intradermally, sublingually, intramuscularly, intraarterially, intravenously, via the central nervous system (CNS, intracerebrally, intracerebroventricularly, intrathecally) or via intraperitoneal administration, particularly epicutaneously, subcutaneously, intradermally, intramuscularly, or intraperitoneally, more particularly subcutaneously, intramuscularly, or intraperitoneally; “formulated to be administered (e.g. intramuscularly)” likewise reads on “formulated for (e.g. intramuscular) administration”.

[0252] In preferred embodiments of the present invention, said pharmaceutical formulation is formulated to be administered or is administered epicutaneously, subcutaneously, intradermally, intramuscularly, intraarterially, intravenously, via the central nervous system (CNS, intracerebrally, intracerebroventricularly, intrathecally) or via intraperitoneal administration, particularly epicutaneously, subcutaneously, intradermally, intramuscularly, or intraperitoneally, more particularly subcutaneously, intramuscularly, or intraperitoneally; “formulated to be administered (e.g. intramuscularly)” likewise reads on “formulated for (e.g. intramuscular) administration.” In the most preferred embodiments of the present invention, said pharmaceutical formulation is formulated to be administered or is administered subcutaneously, intradermally, intramuscularly, intraarterially or intravenously, or via intraperitoneal administration.

[0253] In certain embodiments of the invention the pharmaceutical formulation is to be administered subcutaneously or intravascular (intraarterially, intravenously).

[0254] In certain embodiments of the invention said pharmaceutical formulation is a solution, preferably a ready-to-use solution.

[0255] In certain embodiments of the invention said pharmaceutical formulation is in a freeze-dried state.

[0256] In certain embodiments of the invention said pharmaceutical formulation is administered via infusion.

[0257] In certain specific embodiments of the invention, said pharmaceutical formulation is to be administered systemically.

[0258] In certain specific embodiments of the invention, said pharmaceutical formulation is to be administered subcutaneously.

[0259] According to the present invention the applied dosage might be a single bolus injection delivering the described amount of the compounds to be applied or a continuous infusion of the compounds delivering the desired amount of compounds over a distinct period of time taking into account the velocity of infusion. Thereby the compounds might be applied as a combined injection and / or infusion or as several parallel injections and / or infusion, wherein one or all compounds are delivered as an injection and / or infusions or a distinct compound is injected and another distinct compound is infused.

[0260] Certain embodiments of the present invention relate to the formulation comprising modified or unmodified PAM or fragments thereof and / or optionally one or more pharmaceutically acceptable ingredients.

[0261] Certain embodiments of the present invention relate to the formulation comprising modified or unmodified PAM in combination with ascorbate and / or copper and / or peptide with C-terminal Glycine amino acid or fragments thereof.

[0262] In certain specific embodiments of the invention said peptide with C-terminal Glycine amino acid is selected from the group comprising adrenomedullin (ADM), adrenomedullin-2, intermedin-short, pro-adrenomedullinN-20 terminal peptide (PAMP), amylin, gastrin-releasing peptide, neuromedin C, neuromedin B, neuromedin S, neuromedin U, calcitonin, calcitonin gene-related peptide (CGRP) 1 and 2, islet amyloid polypeptide, chromogranin A, insulin, pancreastatin, prolactin-releasing peptide (PrRP), cholecystokinin, big gastrin, gastrin, glucagon-like peptide 1 (GLP-1), pituitary adenylate cyclase-activating polypeptide (PACAP), secretin, somatoliberin, peptide histidine methionine (PHM), vasoactive intestinal peptide (VIP), gonadoliberin, kisspeptin, MIF-1, metastin, neuropeptide K, neuropeptide Y, neuropeptide gamma, substance P, neurokinin A, neurokinin B, peptide YY, pancreatic hormone, deltorphin I, orexin A and B, melanotropin alpha (alpha-MSH), melanotropin gamma, thyrotropin-releasing hormone (TRH), oxytocin, vasopressin or fragments thereof.

[0263] In certain specific embodiments of the invention said peptide with C-terminal glycine amino acid is selected from the group consisting of adrenomedullin (ADM) vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating polypeptide (PACAP), glucagon-like peptide 1 (GLP-1) and neuropeptide Y or fragments thereof.

[0264] Certain embodiments of the invention relate to a pharmaceutical formulation, wherein said fragments of ADM-Gly (SEQ ID No. 14) are selected from the group of ADM-Gly 2-53 (SEQ ID No. 17), ADM-Gly 8-53 (SEQ ID No. 18), ADM-Gly 22-53 (SEQ ID No. 19), ADM-Gly 27-53 (SEQ ID No. 20) and ADM-Gly 33-53 (SEQ ID No. 21).

[0265] Subject matter of the present invention is a kit comprising the pharmaceutical formulation and one or more items from the group comprising packaging, instructions to the subject and / or physician treating the subject and a leaflet. Such items may in particular comprise instructions regarding the administration of the pharmaceutical formulation to a subject, such as relating to the dosage, medical indications or administration route. More particularly said kit is for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of cerebral blood flow (CBF).

[0266] Dosage

[0267] In certain embodiments of the invention said modified or unmodified PAM or fragments thereof is administered in a dose of 2.0 Units / kg to 116.1 Units / kg, particularly 2.1 Units / kg to 71.5 Units / kg, more particularly 2.2 Units / kg to 54.1 Units / kg, more particularly 2.3 Units / kg to 38.1 Units / kg, more particularly 2.4 Units / kg to 24.1 Units / kg, even more particularly 2.5 Units / kg to 11.1 Units / kg. In certain embodiments of the invention said modified PAM is PEGylated PAM and is administered in a dose of 2.0 Units / kg to 116.1 Units / kg, particularly 2.1 Units / kg to 71.5 Units / kg, more particularly 2.2 Units / kg to 54.1 Units / kg, more particularly 2.3 Units / kg to 38.1 Units / kg, more particularly 2.4 Units / kg to 24.1 Units / kg, even more particularly 2.5 Units / kg to 11.1 Units / kg.

[0268] In certain embodiments of the invention said modified PAM is PEGylated PAM and is administered subcutaneously in a dose of 2.0 Units / kg to 116.1 Units / kg, particularly 2.1 Units / kg to 71.5 Units / kg, more particularly 2.2 Units / kg to 54.1 Units / kg, more particularly 2.3 Units / kg to 38.1 Units / kg, more particularly 2.4 Units / kg to 24.1 Units / kg, even more particularly 2.5 Units / kg to 11.1 Units / kg.

[0269] Certain embodiments of the present invention relate to modified or unmodified Peptidylglycine alpha-amidating monooxygenase (PAM) or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of cerebral blood flow, PAM is in addition to be used with Vitamin C and wherein Vitamin C is applied at a dosage of 1-10000 mg / kg, preferably 2-8000 mg / kg, more preferably 3-6000 mg / kg, more preferably 4-4000 mg / kg, more preferably 5-2000 mg / kg, more preferably 10-1000 mg / kg.

[0270] Certain embodiments of the present invention relate to modified or unmodified Peptidylglycine alpha-amidating monooxygenase (PAM) or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of cerebral blood flow,

[0271] PAM is in addition to be used with Vitamin C and wherein Vitamin C is applied at a dosage of 1-10000 mg / kg, preferably 2-8000 mg / kg, more preferably 3-6000 mg / kg, more preferably 4-4000 mg / kg, more preferably 5-2000 mg / kg, more preferably 10-1000 mg / kg.

[0272] Certain embodiments of the present invention relate to 2-116.1 Units / kg of PAM, particularly 2.1 Units / kg to 71.5 Units / kg, more particularly 2.2 Units / kg to 54.1 Units / kg, more particularly 2.3 Units / kg to 38.1 Units / kg, more particularly 2.4 Units / kg to 24.1 Units / kg, even more particularly 2.5 Units / kg to 11.1 Units / kg are combined with 1-10000 mg / kg, preferably 2-8000 mg / kg, more preferably 3-6000 mg / kg, more preferably 4-4000 mg / kg, more preferably 5-2000 mg / kg, more preferably 10-1000 mg / kg of Vitamin C.

[0273] In certain embodiments of the present invention relate to 2-116.1 Units / kg of PAM are combined with 10-1000 mg / kg of Vitamin C. It is apparent to the skilled person that the above dosages of PAM and / or Vitamin C read equally on the embodiments of the present invention relating to modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF as detailed herein, as well as on embodiments relating to a pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF and likewise on embodiments relating to a kit comprising such pharmaceutical formulation. More particularly said kit is for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF.

[0274] Definitions

[0275] The term “subjects” include human or non-human mammals. Non-human mammals may be for example pigs, dogs, cows, rats, mice, guinea pigs, rabbits, sheep, cats, monkeys, orangutans, or chimpanzees. Preferred are human subjects.

[0276] As used herein, the terms "comprising" and "including" or grammatical variants thereof are to be taken as specifying at least the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof. This term encompasses the terms "consisting of and "consisting essentially of that are understood to specify only the stated feature, integers, steps or components to the exclusion of any additional features.

[0277] Thus, the terms "comprising / including / having" mean that any further component (or likewise features, integers, steps and the like) can / may be present.

[0278] The term "consisting” of means that no further component (or likewise features, integers, steps and the like) is present.

[0279] The term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, biological and biophysical arts.

[0280] The following embodiments form also part of the present invention:

[0281] 1. Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF. Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF according to item 1, wherein said subject has a reduction in CBF.

[0282] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of cerebral blood flow according to item 1 or 2, wherein said subject is suffering from or has a risk of suffering from a disease or medical condition selected from the group comprising a. neuroinflammatory and neurodegenerative diseases; particularly dementia selected from the group comprising MCI, Alzheimer’s disease, vascular dementia, mixed Alzheimer’s disease, and vascular dementia, Lewy body dementia, frontotemporal dementia, focal dementias (including progressive aphasia), subcortical dementias (including Parkinson’s disease), secondary causes of dementia syndrome (including intracranial lesions, multiple sclerosis, Huntington’s Disease, Amyloid Lateral Sclerosis, Traumatic Brain Injury, Creutzfeldt-Jakob Disease);

[0283] b. cardiovascular diseases or conditions, particularly selected from the group comprising atherosclerosis, hypertension, heart failure (including acute and acute decompensated heart failure), atrial fibrillation, cardiovascular ischemia, cerebral ischemic injury, cardiogenic shock, carotid artery stenosis, hypotension, hypertension, peripheral artery disease, coronary artery disease, stroke (including ischemic and haemorrhagic stroke and transient ischemic attack) and myocardial infarction;

[0284] c. metabolic disorders, particularly diabetes mellitus type 1, diabetes mellitus type 2, obesity, and metabolic syndrome;

[0285] d. infections caused by infectious organisms such as bacteria, viruses, fungi or parasites, particularly selected from the group comprising SIRS, sepsis, and septic shock;

[0286] e. respiratory disorders, particularly (chronic obstructive pulmonary disease, sleep apnea)

[0287] f. psychiatric conditions (major depressive disorder, bipolar disorder). Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF according to items 1 to 3, wherein said CBF is assessed with a method selected from the group comprising ultrasound-based techniques for CBF measurements (Transcranial Doppler Ultrasound (TCD); Contrast-Enhanced Ultrasound (CEU), Functional Ultrasound (fUS)), nuclear medicine methods (Single Photon Emission Computed Tomography (SPECT), Positron Emission Tomography (PET)), magnetic resonance imaging (MRI) techniques (Arterial Spin Labeling (ASL), Dynamic Susceptibility Contrast (DSC) MRI, Dynamic Contrast-Enhanced MRI (DCE-MRI), Blood Oxygenation Level-Dependent (BOLD) Functional MRI (fMRI)) and X-ray computed tomography methods (X-Ray Computed Tomography (CT) Perfusion, Xenon-Enhanced CT (Xe-CT)).

[0288] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF, according to items 1 to 4, wherein the CBF is defined as volume of blood expressed in milliliter that passes through 100 g of brain tissue per minute.

[0289] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF, according to items 1 to 5, wherein the cerebral blood flow in said subject is below a certain threshold.

[0290] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF according to item 6, wherein said threshold is defined as a reduction of the CBF below 50 mL / 100g (of brain tissue) per min, preferably below 30 mL / 100g (of brain tissue) per min, more preferably below 15 mL / 100g (of brain tissue) per min and most preferably below 10 mL / 100g (of brain tissue) per min.

[0291] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF according to items 1 to 7, wherein said PAM or fragments thereof is a protein comprising an amino acid sequence selected from the group comprising SEQ ID Nos 1 to 10, or an amino acid sequence having at least 85%, particularly at least 90%, more particularly at least 95%, even more particularly at least 99% sequence identity therewith.

[0292] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF, according to items 1 to 8, wherein said PAM or fragments thereof is enzymatically active.

[0293] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF according to items 1 to 9, wherein said PAM or fragments thereof are modified by d. attaching one or more natural or synthetic polymers units, wherein said polymers are selected from the group comprising

[0294] i. PEG, particularly having an average molecular weight in the range of 0.2-100 kDa, particularly 1-90 kDa, more particularly 2-80 kDa, more particularly 3-70 kDa, more particularly 4-60 kDa and even more particularly 5 to 50kDa; ii. XTEN;

[0295] iii. a peptide polymer consisting of repeats of PAS (the amino acids proline, alanine and serine, particularly comprising 100-200, particularly 120-180, more particularly 130-170, even more particularly about 150 PAS repeats;

[0296] iv. elastin-like polypeptides consisting of Valin-Prolin-Glycine-x-Glycine repeats, wherein x is any amino acid other than Proline, particularly consisting of from 150, 200, 250, 300, 400, or 500 to 550, 600, 750, 850, 950 or 1,000 amino acids; v. HAP polypeptide, having the sequence (Gly4Ser)n, wherein n is 100-200; vi. gelatin-like fusion protein, having the sequence (Gly-X-Y)n, wherein X and Y individually are any amino acid except for Cysteine, and wherein n = 60 to 1500; vii. polysaccharides, in particular those chosen from the group comprising:

[0297] 1. dextrans;

[0298] 2. hydroxyethyls;

[0299] 3. heparosan;

[0300] 4. hyaluronic acid;

[0301] viii. polysialic acid.

[0302] e. conjugation with a serum protein, such as albumin or an immunoglobulin or parts of an immunoglobulin;

[0303] f. site-directed mutagenesis by insertion, deletion and / or exchange of one or more amino acids within the amino acid sequence of PAM or fragments thereof.

[0304] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF according to items 1 to 8, wherein said PAM or fragment thereof is administered in combination with ascorbate and / or copper and / or peptide with C-terminal glycine amino acid or fragments thereof.

[0305] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF according to item 11, wherein said peptide with C-terminal Glycine amino acid is selected from the group of comprising ADM, adrenomedullin-2, intermedin-short, pro-adrenomedullinN-20 terminal peptide (PAMP), amylin, gastrin-releasing peptide, neuromedin C, neuromedin B, neuromedin S, neuromedin U, calcitonin, calcitonin gene-related peptide (CGRP) 1 and 2, islet amyloid polypeptide, chromogranin A, insulin, pancreastatin, prolactin-releasing peptide (PrRP), cholecystokinin, big gastrin, gastrin, glucagon-like peptide 1 (GLP-1), pituitary adenylate cyclase-activating polypeptide (PACAP), secretin, somatoliberin, peptide histidine methionine (PHM), vasoactive intestinal peptide (VIP), gonadoliberin, kisspeptin, MIF-1, metastin, neuropeptide K, neuropeptide gamma, neuropeptide Y, substance P, neurokinin A, neurokinin B, peptide YY, pancreatic hormone, deltorphin I, orexin A and B, melanotropin alpha (alpha-MSH), melanotropin gamma, thyrotropin-releasing hormone (TRH), oxytocin, vasopressin or fragments thereof.

[0306] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF according to items 1 to 12, wherein said subject is characterized by a level of PAM and / or its isoforms and / or fragments thereof below a certain threshold in a bodily fluid of said subject.

[0307] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF according to item 13, wherein said level of PAM and / or its isoforms and / or fragments thereof is the total concentration of PAM or the total activity of PAM.

[0308] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF according to items 13 and 14, wherein the threshold of the total concentration of PAM is equal or below 75 ng / mL, preferably equal or below 65 ng / mL, more preferably equal or below 55 ng / mL, more preferably equal or below 45 ng / mL, more preferably equal or below 35 ng / mL and most preferably equal or below 30 ng / mL.

[0309] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF according to items 13 and 14, wherein the threshold of the activity of PAM is equal or below 11.5 μg / L*h, preferably equal or below 10.5 μg / L*h, more preferably equal or below 9.5 μg / L*h, more preferably equal or below 8.5 μg / L*h, more preferably equal or below 7.5 μg / L*h and most preferably equal or below 6.5 μg / L*h, when PAM activity is measured in serum or wherein the threshold of the activity of PAM equal or below 16.9 μg / L*h, preferably equal or below 15.4 μg / L*h, more preferably equal or below 13.9 μg / L*h, more preferably equal or below 12.3 μg / L*h, more preferably equal or below 10.9 μg / L*h and most preferably equal or below 9.4 μg / L*h, when PAM activity is measured in Li-Heparin.

[0310] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF according to items 13 to 16, wherein the sample of bodily fluid of said subject is selected from the group of whole blood, serum, plasma.

[0311] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF.

[0312] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF according to item 18, wherein said subject has a reduction in cerebral blood-flow.

[0313] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF according to items 18 and 19, wherein said PAM or fragments thereof are isolated from an organism or cell or are recombinantly produced or chimeric PAM or fragments thereof.

[0314] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF according to items 18 to 20, wherein said PAM or a fragment thereof is a protein comprising an amino acid sequence selected from the group comprising SEQ ID Nos 1 to 10, or an amino acid sequence having at least 85%, particularly at least 90%, more particularly at least 95%, even more particularly at least 99% sequence identity therewith.

[0315] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF according to items 18 to 21, wherein said PAM or fragment thereof is enzymatically active.

[0316] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF according to items 18 to 22, wherein said PAM or a fragment thereof is modified by

[0317] d. attaching one or more natural or synthetic polymers units, wherein said polymers are selected from the group comprising

[0318] i. PEG, particularly having an average molecular weight in the range of 0.2-100 kDa, particularly 1-90 kDa, more particularly 2-80 kDa, more particularly 3-70 kDa, more particularly 4-60 kDa and even more particularly 5 to 50kDa; ii. XTEN;

[0319] iii. a peptide polymer consisting of repeats of PAS (the amino acids proline, alanine and serine, particularly comprising 100-200, particularly 120-180, more particularly 130-170, even more particularly about 150 PAS repeats; iv. elastin-like polypeptides consisting of Valin-Prolin-Glycine-x-Glycine repeats, wherein x is any amino acid other than Proline, particularly consisting of from 150, 200, 250, 300, 400, or 500 to 550, 600, 750, 850, 950 or 1,000 amino acids; v. HAP polypeptide, having the sequence (Gly4Ser)n, wherein n is 100-200; vi. gelatin-like fusion protein, having the sequence (Gly-X-Y)n, wherein X and Y individually are any amino acid except for Cysteine, and wherein n = 60 to 1500; vii. polysaccharides, in particular those chosen from the group comprising:

[0320] 1. dextrans;

[0321] 2. hydroxyethyls;

[0322] 3. heparosan;

[0323] 4. hyaluronic acid;

[0324] viii. polysialic acid.

[0325] e. conjugation with a serum protein, such as albumin or an immunoglobulin or parts of an immunoglobulin;

[0326] f. site-directed mutagenesis by insertion, deletion and / or exchange of one or more amino acids within the amino acid sequence of PAM.

[0327] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF according to items 18 to 23, wherein said pharmaceutical formulation is to be administered, epicutaneously, subcutaneously, intradermally, intramuscularly, intravascular (intraarterially, intravenously), or via the central nervous system (CNS, intracerebrally, intracerebroventricularly, intrathecally) or via intraperitoneal administration.

[0328] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF according to items 18 to 24, wherein said pharmaceutical formulation is a solution, preferably a ready -to-use solution.

[0329] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF according to items 18 to 25, wherein said pharmaceutical formulation is in a freeze-dried state. Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF according to items 18 to 26, wherein said pharmaceutical formulation is administered via infusion.

[0330] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF according to items 18 to 27, wherein said pharmaceutical formulation is to be administered systemically.

[0331] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF according to items 18 to 27, wherein said pharmaceutical formulation is to be administered subcutaneously.

[0332] Pharmaceutical formulation according to items 18 to 29, the formulation comprising modified or unmodified PAM or fragments thereof and / or optionally one or more pharmaceutically acceptable ingredients.

[0333] Pharmaceutical formulation modified or unmodified comprising PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF according to items 18 to 30, the formulation comprising said PAM in combination with ascorbate and / or copper and / or peptide with C-terminal Glycine amino acid or fragments thereof.

[0334] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF according to item 31, wherein said peptide with C-terminal Glycine amino acid is selected from the group comprising ADM, adrenomedullin-2, intermedin-short, pro-adrenomedullin N-20 terminal peptide (PAMP), amylin, gastrin-releasing peptide, neuromedin C, neuromedin B, neuromedin S, neuromedin U, calcitonin, calcitonin gene-related peptide (CGRP) 1 and 2, islet amyloid polypeptide, chromogranin A, insulin, pancreastatin, prolactin-releasing peptide (PrRP), cholecystokinin, big gastrin, gastrin, glucagon-like peptide 1 (GLP-1), pituitary adenylate cyclase-activating polypeptide (PACAP), secretin, somatoliberin, peptide histidine methionine (PHM), vasoactive intestinal peptide (VIP), gonadoliberin, kisspeptin, MIF-1, metastin, neuropeptide K, neuropeptide gamma, neuropeptide Y, substance P, neurokinin A, neurokinin B, peptide YY, pancreatic hormone, deltorphin I, orexin A and B, melanotropin alpha (alpha-MSH), melanotropin gamma, thyrotropin-releasing hormone (TRH), oxytocin, vasopressin or fragments thereof.

[0335] 33. A kit comprising the pharmaceutical formulation according to items 18 to 32, more particularly a kit comprising the pharmaceutical formulation according to items 18 to 32 for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF.

[0336] The following embodiments form also part of the present invention:

[0337] 1. Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof whereby the CBF is improved.

[0338] 2. Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof whereby the CBF is improved according to item 1, wherein said subject has a reduction in CBF.

[0339] 3. Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of cerebral blood flow according to item 1 or 2, wherein said subject is suffering from or has a risk of suffering from a disease or medical condition selected from the group comprising a. neuroinflammatory and neurodegenerative diseases; particularly dementia selected from the group comprising MCI, Alzheimer’s disease, vascular dementia, mixed Alzheimer’s disease, and vascular dementia, Lewy body dementia, frontotemporal dementia, focal dementias (including progressive aphasia), subcortical dementias (including Parkinson’s disease), secondary causes of dementia syndrome (including intracranial lesions, multiple sclerosis, Huntington’s Disease, Amyloid Lateral Sclerosis, Traumatic Brain Injury, Creutzfeldt-Jakob Disease); b. cardiovascular diseases or conditions, particularly selected from the group comprising atherosclerosis, hypertension, heart failure (including acute and acute decompensated heart failure), atrial fibrillation, cardiovascular ischemia, cerebral ischemic injury, cardiogenic shock, carotid artery stenosis, hypotension, hypertension, peripheral artery disease, coronary artery disease, stroke (including ischemic and haemorrhagic stroke and transient ischemic attack) and myocardial infarction;

[0340] c. metabolic disorders, particularly diabetes mellitus type 1, diabetes mellitus type 2, obesity, and metabolic syndrome;

[0341] d. infections caused by infectious organisms such as bacteria, viruses, fungi or parasites, particularly selected from the group comprising SIRS, sepsis, and septic shock;

[0342] e. respiratory disorders, particularly (chronic obstructive pulmonary disease, sleep apnea)

[0343] f. psychiatric conditions (major depressive disorder, bipolar disorder).

[0344] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof whereby the CBF is improved according to items 1 to 3, wherein said CBF is assessed with a method selected from the group comprising ultrasound-based techniques for CBF measurements (Transcranial Doppler Ultrasound (TCD); Contrast-Enhanced Ultrasound (CEU), Functional Ultrasound (fUS)), nuclear medicine methods (Single Photon Emission Computed Tomography (SPECT), Positron Emission Tomography (PET)), magnetic resonance imaging (MRI) techniques (Arterial Spin Labeling (ASL), Dynamic Susceptibility Contrast (DSC) MRI, Dynamic Contrast-Enhanced MRI (DCE-MRI), Blood Oxygenation Level-Dependent (BOLD) Functional MRI (fMRI)) and X-ray computed tomography methods (X-Ray Computed Tomography (CT) Perfusion, Xenon-Enhanced CT (Xe-CT)).

[0345] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof whereby the CBF is improved, according to items 1 to 4, wherein the CBF is defined as volume of blood expressed in milliliter that passes through 100 g of brain tissue per minute. Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof whereby the CBF is improved, according to items 1 to 5, wherein the cerebral blood flow in said subject is below a certain threshold.

[0346] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof whereby the CBF is improved according to item 6, wherein said threshold is defined as a reduction of the CBF below 50 mL / 100g (of brain tissue) per min, preferably below 30 mL / 100g (of brain tissue) per min, more preferably below 15 mL / 100g (of brain tissue) per min and most preferably below 10 mL / 100g (of brain tissue) per min.

[0347] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof whereby the CBF is improved according to items 1 to 7, wherein said PAM or fragments thereof is a protein comprising an amino acid sequence selected from the group comprising SEQ ID Nos 1 to 10, or an amino acid sequence having at least 85%, particularly at least 90%, more particularly at least 95%, even more particularly at least 99% sequence identity therewith.

[0348] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof whereby the CBF is improved, according to items 1 to 8, wherein said PAM or fragments thereof is enzymatically active.

[0349] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof whereby the CBF is improved according to items 1 to 9, wherein said PAM or fragments thereof are modified by g. attaching one or more natural or synthetic polymers units, wherein said polymers are selected from the group comprising

[0350] i. PEG, particularly having an average molecular weight in the range of 0.2-100 kDa, particularly 1-90 kDa, more particularly 2-80 kDa, more particularly 3-70 kDa, more particularly 4-60 kDa and even more particularly 5 to 50kDa; ii. XTEN;

[0351] iii. a peptide polymer consisting of repeats of PAS (the amino acids proline, alanine and serine, particularly comprising 100-200, particularly 120-180, more particularly 130-170, even more particularly about 150 PAS repeats;

[0352] iv. elastin-like polypeptides consisting of Valin-Prolin-Glycine-x-Glycine repeats, wherein x is any amino acid other than Proline, particularly consisting of from 150, 200, 250, 300, 400, or 500 to 550, 600, 750, 850, 950 or 1,000 amino acids; v. HAP polypeptide, having the sequence (Gly4Ser)n, wherein n is 100-200; vi. gelatin-like fusion protein, having the sequence (Gly-X-Y)n, wherein X and Y individually are any amino acid except for Cysteine, and wherein n = 60 to 1500; vii. polysaccharides, in particular those chosen from the group comprising:

[0353] 1. dextrans;

[0354] 2. hydroxyethyls;

[0355] 3. heparosan;

[0356] 4. hyaluronic acid;

[0357] viii. polysialic acid.

[0358] h. conjugation with a serum protein, such as albumin or an immunoglobulin or parts of an immunoglobulin;

[0359] i. site-directed mutagenesis by insertion, deletion and / or exchange of one or more amino acids within the amino acid sequence of PAM or fragments thereof.

[0360] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof whereby the cerebral blood flow (CBF) is improved according to items 1 to 8, wherein said PAM or fragment thereof is administered in combination with ascorbate and / or copper and / or peptide with C-terminal glycine amino acid or fragments thereof.

[0361] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof whereby the cerebral blood flow (CBF) is improved according to item 11, wherein said peptide with C-terminal Glycine amino acid is selected from the group of comprising adrenomedullin (ADM), adrenomedullin-2, intermedin-short, pro-adrenomedullin N-20 terminal peptide (PAMP), amylin, gastrin-releasing peptide, neuromedin C, neuromedin B, neuromedin S, neuromedin U, calcitonin, calcitonin gene-related peptide (CGRP) 1 and 2, islet amyloid polypeptide, chromogranin A, insulin, pancreastatin, prolactin-releasing peptide (PrRP), cholecystokinin, big gastrin, gastrin, glucagon-like peptide 1 (GLP-1), pituitary adenylate cyclase-activating polypeptide (PACAP), secretin, somatoliberin, peptide histidine methionine (PHM), vasoactive intestinal peptide (VIP), gonadoliberin, kisspeptin, MIF-1, metastin, neuropeptide K, neuropeptide gamma, neuropeptide Y, substance P, neurokinin A, neurokinin B, peptide YY, pancreatic hormone, deltorphin I, orexin A and B, melanotropin alpha (alpha-MSH), melanotropin gamma, thyrotropin-releasing hormone (TRH), oxytocin, vasopressin or fragments thereof.

[0362] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof whereby the CBF is improved according to items 1 to 12, wherein said subject is characterized by a level of PAM and / or its isoforms and / or fragments thereof below a certain threshold in a bodily fluid of said subject.

[0363] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof whereby the CBF is improved according to item 13, wherein said level of PAM and / or its isoforms and / or fragments thereof is the total concentration of PAM or the total activity of PAM.

[0364] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof whereby the CBF is improved according to items 13 and 14, wherein the threshold of the total concentration of PAM is equal or below 75 ng / mL, preferably equal or below 65 ng / mL, more preferably equal or below 55 ng / mL, more preferably equal or below 45 ng / mL, more preferably equal or below 35 ng / mL and most preferably equal or below 30 ng / mL.

[0365] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof whereby the CBF is improved according to items 13 and 14, wherein the threshold of the activity of PAM is equal or below 11.5 μg / L*h, preferably equal or below 10.5 μg / L*h, more preferably equal or below 9.5 μg / L*h, more preferably equal or below 8.5 μg / L*h, more preferably equal or below 7.5 μg / L*h and most preferably equal or below 6.5 μg / L*h, when PAM activity is measured in serum or wherein the threshold of the activity of PAM equal or below 16.9 pg / L*h, preferably equal or below 15.4 pg / L*h, more preferably equal or below 13.9 pg / L*h, more preferably equal or below 12.3 pg / L*h, more preferably equal or below 10.9 μg / L*h and most preferably equal or below 9.4 μg / L*h, when PAM activity is measured in Li-Heparin.

[0366] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof whereby the CBF is improved according to items 13 to 16, wherein the sample of bodily fluid of said subject is selected from the group of whole blood, serum, plasma.

[0367] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof whereby the CBF is improved.

[0368] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof whereby the CBF is improved according to item 18, wherein said subject has a reduction in CBF.

[0369] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof whereby the CBF is improved according to items 18 and 19, wherein said PAM or fragments thereof are isolated from an organism or cell or are recombinantly produced or chimeric PAM or fragments thereof.

[0370] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof whereby the CBF is improved according to items 18 to 20, wherein said PAM or a fragment thereof is a protein comprising an amino acid sequence selected from the group comprising SEQ ID Nos 1 to 10, or an amino acid sequence having at least 85%, particularly at least 90%, more particularly at least 95%, even more particularly at least 99% sequence identity therewith.

[0371] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof whereby the CBF is improved according to items 18 to 21, wherein said PAM or fragment thereof is enzymatically active.

[0372] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof whereby the CBF is improved according to items 18 to 22, wherein said PAM or a fragment thereof is modified by

[0373] g. attaching one or more natural or synthetic polymers units, wherein said polymers are selected from the group comprising

[0374] i. PEG, particularly having an average molecular weight in the range of 0.2-100 kDa, particularly 1-90 kDa, more particularly 2-80 kDa, more particularly 3-70 kDa, more particularly 4-60 kDa and even more particularly 5 to 50kDa; ii. XTEN;

[0375] iii. a peptide polymer consisting of repeats of PAS (the amino acids proline, alanine and serine, particularly comprising 100-200, particularly 120-180, more particularly 130-170, even more particularly about 150 PAS repeats;

[0376] iv. elastin-like polypeptides consisting of Valin-Prolin-Glycine-x-Glycine repeats, wherein x is any amino acid other than Proline, particularly consisting of from 150, 200, 250, 300, 400, or 500 to 550, 600, 750, 850, 950 or 1,000 amino acids; v. HAP polypeptide, having the sequence (Gly4Ser)n, wherein n is 100-200; vi. gelatin-like fusion protein, having the sequence (Gly-X-Y)n, wherein X and Y individually are any amino acid except for Cysteine, and wherein n = 60 to 1500; vii. polysaccharides, in particular those chosen from the group comprising:

[0377] 1. dextrans;

[0378] 2. hydroxyethyls;

[0379] 3. heparosan;

[0380] 4. hyaluronic acid;

[0381] viii. polysialic acid.

[0382] h. conjugation with a serum protein, such as albumin or an immunoglobulin or parts of an immunoglobulin;

[0383] i. site-directed mutagenesis by insertion, deletion and / or exchange of one or more amino acids within the amino acid sequence of PAM. Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof whereby the CBF is improved according to items 18 to 23, wherein said pharmaceutical formulation is to be administered, epicutaneously, subcutaneously, intradermally, intramuscularly, intravascular (intraarterially, intravenously), or via the central nervous system (CNS, intracerebrally, intracerebroventricularly, intrathecally) or via intraperitoneal administration.

[0384] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof whereby the CBF is improved according to items 18 to 24, wherein said pharmaceutical formulation is a solution, preferably a ready-to-use solution.

[0385] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof whereby the CBF is improved according to items 18 to 25, wherein said pharmaceutical formulation is in a freeze-dried state.

[0386] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof whereby the CBF is improved according to items 18 to 26, wherein said pharmaceutical formulation is administered via infusion.

[0387] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof whereby the CBF is improved according to items 18 to 27, wherein said pharmaceutical formulation is to be administered systemically.

[0388] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof whereby the CBF is improved according to items 18 to 27, wherein said pharmaceutical formulation is to be administered subcutaneously. 30. Pharmaceutical formulation according to items 18 to 29, the formulation comprising modified or unmodified PAM or fragments thereof and / or optionally one or more pharmaceutically acceptable ingredients.

[0389] 31. Pharmaceutical formulation modified or unmodified comprising PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof whereby the CBF is improved according to items 18 to 30, the formulation comprising said PAM in combination with ascorbate and / or copper and / or peptide with C-terminal Glycine amino acid or fragments thereof.

[0390] 32. Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof whereby the CBF is improved according to item 31, wherein said peptide with C-terminal Glycine amino acid is selected from the group comprising ADM, adrenomedullin-2, intermedin-short, pro-adrenomedullin N-20 terminal peptide (PAMP), amylin, gastrin-releasing peptide, neuromedin C, neuromedin B, neuromedin S, neuromedin U, calcitonin, calcitonin gene-related peptide (CGRP) 1 and 2, islet amyloid polypeptide, chromogranin A, insulin, pancreastatin, prolactin-releasing peptide (PrRP), cholecystokinin, big gastrin, gastrin, glucagon-like peptide 1 (GLP-1), pituitary adenylate cyclase-activating polypeptide (PACAP), secretin, somatoliberin, peptide histidine methionine (PHM), vasoactive intestinal peptide (VIP), gonadoliberin, kisspeptin, MIF-1, metastin, neuropeptide K, neuropeptide gamma, neuropeptide Y, substance P, neurokinin A, neurokinin B, peptide YY, pancreatic hormone, deltorphin I, orexin A and B, melanotropin alpha (alpha-MSH), melanotropin gamma, thyrotropinreleasing hormone (TRH), oxytocin, vasopressin or fragments thereof.

[0391] 33. A kit comprising the pharmaceutical formulation according to items 18 to 32, more particularly a kit comprising the pharmaceutical formulation according to items 18 to 32 for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF.

[0392] The embodiments of the present invention relating to modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF likewise relate to the use of modified or unmodified PAM or fragments thereof in the production of a medicament for the for the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF, the use of modified or unmodified PAM or fragments thereof for the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF, as well as methods of treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF comprising the administration of modified or unmodified PAM or fragments thereof to a subject in need thereof. Mutatis mutandis, this relates to the corresponding pharmaceutical formulations.

[0393] The present invention is further described by reference to the following non-limiting figures.

[0394] Brief description of figures

[0395] Fig- 1: Schematic representation of PAM Isoform 1. Black bold arrows indicate cleavage-sites at double-basic amino-acids.

[0396] Fig 2: Representative enzymatic reaction catalysed by PAM.

[0397] Fig 3: A representative SDS-PAGE of recombinant human PAM after and before PEGylation.

[0398] Fig 4A: Representative calibration curve of PAM Amidating Activity Assay (PAM- AMA). Fig 4B: Frequency distribution (histogram) of PAM- AMA in self-reported healthy individuals (n=120)

[0399] Fig4C: Correlation of PAM- AMA in matrix duplets (Li-heparin and serum) from self-reported healthy individuals (n=20)

[0400] Fig- 5: Enhancement of PAM activity in circulation after intravenous administration of single PHM subunit, or in combination with PAL subunit.

[0401] Fig.6A: Cerebral blood flow in cortex at a baseline prior ischemia induction in stoke and sham animals

[0402] Fig. 6B: Intraperitoneal administration of PAM restores cortical cerebral blood flow postischemia

[0403] Fig. 6C: Cerebral blood flow in striatum at a baseline prior ischemia induction in stoke and sham animals Fig. 6D: Intraperitoneal administration of PAM restores striatal cerebral blood flow postischemia

[0404] Fig. 7A: Intraperitoneal administration of PAM improves capillary function in cortex in postischemia as shown by ex vivo fluorescent vascular cast technique

[0405] Fig. 7B: Intraperitoneal administration of PAM improves capillary function in cortex in postischemia based on the decreased laminin-to-infarct volume ratio

[0406] Fig. 8A-F: Intraperitoneal administration of PAM reduces reactive astrocytes in dentate gyrus (A), stratum lacunosum-moleculare (B), CA1 region of hippocampus (C), CA2 region of hippocampus (D), CA3 region of hippocampus (E) and total hippocampus (F).

[0407] Fig- 9: Left: comparative activity analysis of pegylated or xtenylated (i.e. modified with XTEN) and unmodified PAM enzymes. Right: comparative analysis of pegylated or xtenylated and unmodified PAM enzymes on an SDS-PAGE

[0408] Sequences

[0409] SEQ ID NO: 1 - PAM isoform 1 AS 31-973 of Prepro-PAM Isoform 1 (amino acids 1-30 of SEQ ID No. 30 missing)

[0410] 10 20 30 40 50 FKETTRPFSN ECLGTTRPVV PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR

[0411] 60 70 80 90 100 IPVDEEAFVI DFKPRASMDT VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA

[0412] 110 120 130 140 150 NILYAWARNA PPTRLPKGVG FRVGGETGSK YFVLQVHYGD ISAFRDNNKD

[0413] 160 170 180 190 200 CSGVSLHLTR LPQPLIAGMY LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH

[0414] 210 220 230 240 250 VFAYRVHTHH LGKVVSGYRV RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF

[0415] 260 270 280 290 300 GDLLAARCVF TGEGRTEATH IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT

[0416] 310 320 330 340 350 QNVAPDMFRT IPPEANIPIP VKSDMVMMHE HHKETEYKDK IPLLQQPKRE

[0417] 360 370 380 390 400 EEEVLDQGDF YSLLSKLLGE REDVVHVHKY NPTEKAESES DLVAEIANW

[0418] 410 420 430 440 450 QKKDLGRSDA REGAEHERGN AILVRDRIHK FHRLVSTLRP PESRVFSLQQ

[0419] 460 470 480 490 500 PPPGEGTWEP EHTGDFHMEE ALDWPGVYLL PGQVSGVALD PKNNLVIFHR

[0420] 510 520 530 540 550 GDHVWDGNSF DSKFVYQQIG LGPIEEDTIL VIDPNNAAVL QSSGKNLFYL

[0421] 560 570 580 590 600 PHGLSIDKDG NYWVTDVALH QVFKLDPNNK EGPVLILGRS MQPGSDQNHF

[0422] 610 620 630 640 650 CQPTDVAVDP GTGAIYVSDG YCNSRIVQFS PSGKFITQWG EESSGSSPLP 660 670 680 690 700 GQFTVPHSLA LVPLLGQLCV ADRENGRIQC FKTDTKEFVR EIKHSSFGRN

[0423] 710 720 730 740 750 VFAISYIPGL LFAVNGKPHF GDQEPVQGFV MNFSNGEIID IFKPVRKHFD

[0424] 760 770 780 790 800 MPHDIVASED GTVYIGDAHT NTVWKFTLTE KLEHRSVKKA GIEVQEIKEA

[0425] 810 820 830 840 850 EAVVETKMEN KPTSSELQKM QEKQKLIKEP GSGVPVVLIT TLLVIPVVVL

[0426] 860 870 880 890 900 LAIAIFIRWK KSRAFGDSEH KLETSSGRVL GRFRGKGSGG LNLGNFFASR

[0427] 910 920 930 940 943

[0428] KGYSRKGFDR LSTEGSDQEK EDDGSESEEE YSAPLPALAP SSS

[0429] SEQ ID NO: 2 - PAM isoform 2 AS 31-868 of prepro-PAM isoform 2 (amino acids 1-30 of SEQ ID No. 31 missing)

[0430] 10 20 30 40 50 FKETTRPFSN ECLGTTRPVV PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR

[0431] 60 70 80 90 100 IPVDEEAFVI DFKPRASMDT VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA

[0432] 110 120 130 140 150 NILYAWARNA PPTRLPKGVG FRVGGETGSK YFVLQVHYGD ISAFRDNNKD

[0433] 160 170 180 190 200 CSGVSLHLTR LPQPLIAGMY LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH

[0434] 210 220 230 240 250 VFAYRVHTHH LGKVVSGYRV RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF

[0435] 260 270 280 290 300 GDLLAARCVF TGEGRTEATH IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT

[0436] 310 320 330 340 350 QNVAPDMFRT IPPEANIPIP VKSDMVMMHE HHKETEYKDK IPLLQQPKRE

[0437] 360 370 380 390 400 EEEVLDQDFH MEEALDWPGV YLLPGQVSGV ALDPKNNLVI FHRGDHVWDG

[0438] 410 420 430 440 450 NSFDSKFVYQ QIGLGPIEED TILVIDPNNA AVLQSSGKNL FYLPHGLSID

[0439] 460 470 480 490 500 KDGNYWVTDV ALHQVFKLDP NNKEGPVLIL GRSMQPGSDQ NHFCQPTDVA

[0440] 510 520 530 540 550 VDPGTGAIYV SDGYCNSRIV QFSPSGKFIT QWGEESSGSS PLPGQFTVPH

[0441] 560 570 580 590 600 SLALVPLLGQ LCVADRENGR IQCFKTDTKE FVREIKHSSF GRNVFAISYI

[0442] 610 620 630 640 650 PGLLFAVNGK PHFGDQEPVQ GFVMNFSNGE IIDIFKPVRK HFDMPHDIVA

[0443] 660 670 680 690 700 SEDGTVYIGD AHTNTVWKFT LTEKLEHRSV KKAGIEVQEI KEAEAWETK

[0444] 710 720 730 740 750 MENKPTSSEL QKMQEKQKLI KEPGSGVPVV LITTLLVIPV WLLAIAIFI

[0445] 760 770 780 790 800 RWKKSRAFGD SEHKLETSSG RVLGRFRGKG SGGLNLGNFF ASRKGYSRKG

[0446] 810 820 830 836

[0447] FDRLSTEGSD QEKEDDGSES EEEYSAPLPA LAPSSS SEQ ID No: 3 - PAM isoform 3 AS 31-905 of prepro-PAM isoform 3 (amino acids 1-30 and 829-896 of SEQ ID No. 30 missing)

[0448] 10 20 30 40 50 FKETTRPFSN ECLGTTRPVV PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR

[0449] 60 70 80 90 100 IPVDEEAFVI DFKPRASMDT VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA

[0450] 110 120 130 140 150 NILYAWARNA PPTRLPKGVG FRVGGETGSK YFVLQVHYGD ISAFRDNNKD

[0451] 160 170 180 190 200 CSGVSLHLTR LPQPLIAGMY LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH

[0452] 210 220 230 240 250 VFAYRVHTHH LGKVVSGYRV RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF

[0453] 260 270 280 290 300 GDLLAARCVF TGEGRTEATH IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT

[0454] 310 320 330 340 350 QNVAPDMFRT IPPEANIPIP VKSDMVMMHE HHKETEYKDK IPLLQQPKRE

[0455] 360 370 380 390 400 EEEVLDQGDF YSLLSKLLGE REDVVHVHKY NPTEKAESES DLVAEIANW

[0456] 410 420 430 440 450 QKKDLGRSDA REGAEHERGN AILVRDRIHK FHRLVSTLRP PESRVFSLQQ

[0457] 460 470 480 490 500 PPPGEGTWEP EHTGDFHMEE ALDWPGVYLL PGQVSGVALD PKNNLVIFHR

[0458] 510 520 530 540 550 GDHVWDGNSF DSKFVYQQIG LGPIEEDTIL VIDPNNAAVL QSSGKNLFYL

[0459] 560 570 580 590 600 PHGLSIDKDG NYWVTDVALH QVFKLDPNNK EGPVLILGRS MQPGSDQNHF

[0460] 610 620 630 640 650 CQPTDVAVDP GTGAIYVSDG YCNSRIVQFS PSGKFITQWG EESSGSSPLP

[0461] 660 670 680 690 700 GQFTVPHSLA LVPLLGQLCV ADRENGRIQC FKTDTKEFVR EIKHSSFGRN

[0462] 710 720 730 740 750 VFAISYIPGL LFAVNGKPHF GDQEPVQGFV MNFSNGEIID IFKPVRKHFD

[0463] 760 770 780 790 800 MPHDIVASED GTVYIGDAHT NTVWKFTLTE KLEHRSVKKA GIEVQEIKDS

[0464] 810 820 830 840 850 EHKLETSSGR VLGRFRGKGS GGLNLGNFFA SRKGYSRKGF DRLSTEGSDQ

[0465] 860 870 885

[0466] EKEDDGSESE EEYSAPLPAL APSSS SEQ ID No: 4 - PAM isoform 4 AS 31-887 of prepro-PAM isoform 4 (amino acids 1-30 and 829-914 of SEQ ID No. 30 missing)

[0467] 10 20 30 40 50 FKETTRPFSN ECLGTTRPVV PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR

[0468] 60 70 80 90 100 IPVDEEAFVI DFKPRASMDT VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA

[0469] 110 120 130 140 150 NILYAWARNA PPTRLPKGVG FRVGGETGSK YFVLQVHYGD ISAFRDNNKD

[0470] 160 170 180 190 200 CSGVSLHLTR LPQPLIAGMY LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH

[0471] 210 220 230 240 250 VFAYRVHTHH LGKVVSGYRV RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF 260 270 280 290 300 GDLLAARCVF TGEGRTEATH IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT

[0472] 310 320 330 340 350 QNVAPDMFRT IPPEANIPIP VKSDMVMMHE HHKETEYKDK IPLLQQPKRE

[0473] 360 370 380 390 400 EEEVLDQGDF YSLLSKLLGE REDVVHVHKY NPTEKAESES DLVAEIANW

[0474] 410 420 430 440 450 QKKDLGRSDA REGAEHERGN AILVRDRIHK FHRLVSTLRP PESRVFSLQQ

[0475] 460 470 480 490 500 PPPGEGTWEP EHTGDFHMEE ALDWPGVYLL PGQVSGVALD PKNNLVIFHR

[0476] 510 520 530 540 550 GDHVWDGNSF DSKFVYQQIG LGPIEEDTIL VIDPNNAAVL QSSGKNLFYL

[0477] 560 570 580 590 600 PHGLSIDKDG NYWVTDVALH QVFKLDPNNK EGPVLILGRS MQPGSDQNHF

[0478] 610 620 630 640 650 CQPTDVAVDP GTGAIYVSDG YCNSRIVQFS PSGKFITQWG EESSGSSPLP

[0479] 660 670 680 690 700 GQFTVPHSLA LVPLLGQLCV ADRENGRIQC FKTDTKEFVR EIKHSSFGRN

[0480] 710 720 730 740 750 VFAISYIPGL LFAVNGKPHF GDQEPVQGFV MNFSNGEIID IFKPVRKHFD

[0481] 760 770 780 790 800 MPHDIVASED GTVYIGDAHT NTVWKFTLTE KLEHRSVKKA GIEVQEIKGK

[0482] 810 820 830 840 850 GSGGLNLGNF FASRKGYSRK GFDRLSTEGS DQEKEDDGSE SEEEYSAPLP

[0483] 857

[0484] ALAPSSS SEQ ID No: 5 - PAM Isoform 5 AS 31-973 of prepro-PAM Isoform 5 (SEQ ID No. 30 with an additional aa in position 896 and amino acids 1-30 missing)

[0485] 10 20 30 40 50 FKETTRPFSN ECLGTTRPVV PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR

[0486] 60 70 80 90 100 IPVDEEAFVI DFKPRASMDT VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA

[0487] 110 120 130 140 150 NILYAWARNA PPTRLPKGVG FRVGGETGSK YFVLQVHYGD ISAFRDNNKD

[0488] 160 170 180 190 200 CSGVSLHLTR LPQPLIAGMY LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH

[0489] 210 220 230 240 250 VFAYRVHTHH LGKVVSGYRV RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF

[0490] 260 270 280 290 300 GDLLAARCVF TGEGRTEATH IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT

[0491] 310 320 330 340 350 QNVAPDMFRT IPPEANIPIP VKSDMVMMHE HHKETEYKDK IPLLQQPKRE

[0492] 360 370 380 390 400 EEEVLDQGDF YSLLSKLLGE REDVVHVHKY NPTEKAESES DLVAEIANW

[0493] 410 420 430 440 450 QKKDLGRSDA REGAEHERGN AILVRDRIHK FHRLVSTLRP PESRVFSLQQ

[0494] 460 470 480 490 500 PPPGEGTWEP EHTGDFHMEE ALDWPGVYLL PGQVSGVALD PKNNLVIFHR

[0495] 510 520 530 540 550 GDHVWDGNSF DSKFVYQQIG LGPIEEDTIL VIDPNNAAVL QSSGKNLFYL 560 570 580 590 600 PHGLSIDKDG NYWVTDVALH QVFKLDPNNK EGPVLILGRS MQPGSDQNHF

[0496] 610 620 630 640 650 CQPTDVAVDP GTGAIYVSDG YCNSRIVQFS PSGKFITQWG EESSGSSPLP

[0497] 660 670 680 690 700 GQFTVPHSLA LVPLLGQLCV ADRENGRIQC FKTDTKEFVR EIKHSSFGRN

[0498] 710 720 730 740 750 VFAISYIPGL LFAVNGKPHF GDQEPVQGFV MNFSNGEIID IFKPVRKHFD

[0499] 760 770 780 790 800 MPHDIVASED GTVYIGDAHT NTVWKFTLTE KLEHRSVKKA GIEVQEIKEA

[0500] 810 820 830 840 850 EAVVETKMEN KPTSSELQKM QEKQKLIKEP GSGVPVVLIT TLLVIPVVVL

[0501] 860 870 880 890 900

[0502] LAIAI FIRWK KSRAFGADSE HKLETSSGRV LGRFRGKGSG GLNLGNFFAS

[0503] 910 920 930 940 944

[0504] RKGYSRKGFD RLSTEGSDQE KEDDGSESEE EYSAPLPALA PSSS SEQ ID No: 6 - PAM Isoform 6 AS 31-955 of prepro-PAM Isoform 6 (amino acids 1-30 and 897-914 of SEQ ID No. 30 missing)

[0505] 10 20 30 40 50 FKETTRPFSN ECLGTTRPVV PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR

[0506] 60 70 80 90 100 IPVDEEAFVI DFKPRASMDT VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA

[0507] 110 120 130 140 150 NILYAWARNA PPTRLPKGVG FRVGGETGSK YFVLQVHYGD ISAFRDNNKD

[0508] 160 170 180 190 200 CSGVSLHLTR LPQPLIAGMY LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH

[0509] 210 220 230 240 250 VFAYRVHTHH LGKVVSGYRV RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF

[0510] 260 270 280 290 300 GDLLAARCVF TGEGRTEATH IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT

[0511] 310 320 330 340 350 QNVAPDMFRT IPPEANIPIP VKSDMVMMHE HHKETEYKDK IPLLQQPKRE

[0512] 360 370 380 390 400 EEEVLDQGDF YSLLSKLLGE REDVVHVHKY NPTEKAESES DLVAEIANW

[0513] 410 420 430 440 450 QKKDLGRSDA REGAEHERGN AILVRDRIHK FHRLVSTLRP PESRVFSLQQ

[0514] 460 470 480 490 500 PPPGEGTWEP EHTGDFHMEE ALDWPGVYLL PGQVSGVALD PKNNLVIFHR

[0515] 510 520 530 540 550 GDHVWDGNSF DSKFVYQQIG LGPIEEDTIL VIDPNNAAVL QSSGKNLFYL

[0516] 560 570 580 590 600 PHGLSIDKDG NYWVTDVALH QVFKLDPNNK EGPVLILGRS MQPGSDQNHF

[0517] 610 620 630 640 650 CQPTDVAVDP GTGAIYVSDG YCNSRIVQFS PSGKFITQWG EESSGSSPLP

[0518] 660 670 680 690 700 GQFTVPHSLA LVPLLGQLCV ADRENGRIQC FKTDTKEFVR EIKHSSFGRN

[0519] 710 720 730 740 750 VFAISYIPGL LFAVNGKPHF GDQEPVQGFV MNFSNGEIID IFKPVRKHFD

[0520] 760 770 780 790 800 MPHDIVASED GTVYIGDAHT NTVWKFTLTE KLEHRSVKKA GIEVQEIKEA

[0521] 810 820 830 840 850 EAVVETKMEN KPTSSELQKM QEKQKLIKEP GSGVPVVLIT TLLVIPVVVL 860 870 880 890 900 LAIAI FIRWK KSRAFGGKGS GGLNLGNFFA SRKGYSRKGF DRLSTEGSDQ

[0522] 910 920 925

[0523] EKEDDGSESE EEYSAPLPAL APSSS SEQ ID No: 7 - PHM subunit of PAM

[0524] 10 20 30 40 50 FKETTRPFSN ECLGTTRPVV PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR

[0525] 60 70 80 90 100 IPVDEEAFVI DFKPRASMDT VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA

[0526] 110 120 130 140 150 NILYAWARNA PPTRLPKGVG FRVGGETGSK YFVLQVHYGD ISAFRDNNKD

[0527] 160 170 180 190 200 CSGVSLHLTR LPQPLIAGMY LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH

[0528] 210 220 230 240 250 VFAYRVHTHH LGKVVSGYRV RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF

[0529] 260 270 280 290 300 GDLLAARCVF TGEGRTEATH IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT

[0530] 310 320 330 340 350 QNVAPDMFRT IPPEANIPIP VKSDMVMMHE HHKETEYKDK IPLLQQPKRE

[0531] 360 370 380 390 400 EEEVLDQGDF YSLLSKLLGE REDVVHVHKY NPTEKAESES DLVAEIANW

[0532] 410 420 430 440 450 QKKDLGRSDA REGAEHERGN AILVRDRIHK FHRLVSTLRP PESRVFSLQQ

[0533] 460

[0534] PPPGEGTWEP EHTG SEQ ID No: 8 - PAL subunit of PAM

[0535] 10 20 30 40 50 DFHMEEALDW PGVYLLPGQV SGVALDPKNN LVIFHRGDHV WDGNSFDSKF

[0536] 60 70 80 90 100 VYQQIGLGPI EEDTILVIDP NNAAVLQSSG KNLFYLPHGL SIDKDGNYWV

[0537] 110 120 130 140 150 TDVALHQVFK LDPNNKEGPV LILGRSMQPG SDQNHFCQPT DVAVDPGTGA

[0538] 160 170 180 190 200 IYVSDGYCNS RIVQFSPSGK FITQWGEESS GSSPLPGQFT VPHSLALVPL

[0539] 210 220 230 240 250 LGQLCVADRE NGRIQCFKTD TKEFVREIKH SSFGRNVFAI SYIPGLLFAV

[0540] 260 270 280 290 300 NGKPHFGDQE PVQGFVMNFS NGEIIDIFKP VRKHFDMPHD IVASEDGTVY

[0541] 310 320

[0542] IGDAHTNTVW KFTLTEKLEH RSV

[0543] SEQ ID No. 9 - PHM fragment (aa 31-377 of PAM SEQ ID No. 1)

[0544] 10 20 30 40 50 FKETTRPFSN ECLGTTRPVV PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR

[0545] 60 70 80 90 100 IPVDEEAFVI DFKPRASMDT VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA

[0546] 110 120 130 140 150 NILYAWARNA PPTRLPKGVG FRVGGETGSK YFVLQVHYGD ISAFRDNNKD 160 170 180 190 200 CSGVSLHLTR LPQPLIAGMY LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH

[0547] 210 220 230 240 250 VFAYRVHTHH LGKVVSGYRV RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF

[0548] 260 270 280 290 300 GDLLAARCVF TGEGRTEATH IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT

[0549] 310 320 330 340 347 QNVAPDMFRT IPPEANIPIP VKSDMVMMHE HHKETEYKDK IPLLQQP SEQ ID No: 10 - Sequence of recombinant human PAM

[0550] 10 20 30 40 50 SPLSVFKRFK ETTRPFSNEC LGTTRPWPI DSSDFALDIR MPGVTPKQSD

[0551] 60 70 80 90 100 TYFCMSMRIP VDEEAFVIDF KPRASMDTVH HMLLFGCNMP SSTGSYWFCD

[0552] 110 120 130 140 150 EGTCTDKANI LYAWARNAPP TRLPKGVGFR VGGETGSKYF VLQVHYGDIS

[0553] 160 170 180 190 200 AFRDNNKDCS GVSLHLTRLP QPLIAGMYLM MSVDTVIPAG EKWNSDISC

[0554] 210 220 230 240 250 HYKNYPMHVF AYRVHTHHLG KWSGYRVRN GQWTLIGRQS PQLPQAFYPV

[0555] 260 270 280 290 300 GHPVDVSFGD LLAARCVFTG EGRTEATHIG GTSSDEMCNL YIMYYMEAKH

[0556] 310 320 330 340 350 AVSFMTCTQN VAPDMFRTIP PEANIPIPVK SDMVMMHEHH KETEYKDKIP

[0557] 360 370 380 390 400 LLQQPKREEE EVLDQGDFYS LLSKLLGERE DWHVHKYNP TEKAESESDL

[0558] 410 420 430 440 450 VAEIANWQK KDLGRSDARE GAEHERGNAI LVRDRIHKFH RLVSTLRPPE

[0559] 460 470 480 490 500 SRVFSLQQPP PGEGTWEPEH TGDFHMEEAL DWPGVYLLPG QVSGVALDPK

[0560] 510 520 530 540 550 NNLVIFHRGD HVWDGNSFDS KFVYQQIGLG PIEEDTILVI DPNNAAVLQS

[0561] 560 570 580 590 600 SGKNLFYLPH GLSIDKDGNY WVTDVALHQV FKLDPNNKEG PVLILGRSMQ

[0562] 610 620 630 640 650 PGSDQNHFCQ PTDVAVDPGT GAIYVSDGYC NSRIVQFSPS GKFITQWGEE

[0563] 660 670 680 690 700 SSGSSPLPGQ FTVPHSLALV PLLGQLCVAD RENGRIQCFK TDTKEFVREI

[0564] 710 720 730 740 750 KHSSFGRNVF AISYIPGLLF AVNGKPHFGD QEPVQGFVMN FSNGEIIDIF

[0565] 760 770 780 790 800 KPVRKHFDMP HDIVASEDGT VYIGDAHTNT VWKFTLTEKL EHRSVKKAGI

[0566] 810

[0567] EVQEIKEAEA WGS SEQ ID No: 11 - pre-proADM (aa 1-185)

[0568] 10 20 30 40 50 MKLVSVALMY LGSLAFLGAD TARLDVASEF RKKWNKWALS RGKRELRMSS

[0569] 60 70 80 90 100 SYPTGLADVK AGPAQTLIRP QDMKGASRSP EDSSPDAARI RVKRYRQSMN

[0570] 110 120 130 140 150 NFQGLRSFGC RFGTCTVQKL AHQIYQFTDK DKDNVAPRSK ISPQGYGRRR 160 170 180

[0571] RRSLPEAGPG RTLVSSKPQA HGAPAPPSGS APHFL SEQ ID No: 12 - PAMP-Gly (aa 22-42 of pre-proADM SEQ ID No. 11)

[0572] 10 20

[0573] ARLDVASEFR KKWNKWALSR G SEQ ID No: 13 - CT-proADM (aa 148-185 of pre-proADM SEQ ID No. 11)

[0574] 10 20 30

[0575] RRRRRSLPEA GPGRTLVSSK PQAHGAPAPP SGSAPHFL SEQ ID No: 14 - ADM-Gly (aa 95-147 of pre-proADM SEQ ID No. 11)

[0576] 10 20 30 40 50 YRQSMNNFQG LRSFGCRFGT CTVQKLAHQI YQFTDKDKDN VAPRSKISPQ GYG SEQ ID No: 15 - bio-ADM (aa 95-146 of pre-proADM SEQ ID No. 11)

[0577] 10 20 30 40 50 YRQSMNNFQG LRSFGCRFGT CTVQKLAHQI YQFTDKDKDN VAPRSKISPQ GY-CONH2SEQ ID No: 16 - MR-proADM (aa 45-92 of pre-proADM SEQ ID No. 11)

[0578] 10 20 30 40

[0579] ELRMSSSYPT GLADVKAGPA QTLIRPQDMK GASRSPEDSS PDAARIRV SEQ ID No: 17 - ADM-Gly 2-53 (aa 96-147 of pre-proADM SEQ ID No. 11)

[0580] 10 20 30 40 50 RQSMNNFQGL RSFGCRFGTC TVQKLAHQIY QFTDKDKDNV APRSKISPQG YG SEQ ID No: 18 - ADM-Gly 8-53 (aa 102-147 of pre-proADM SEQ ID No. 11)

[0581] 10 20 30 40 FQGLRSFGCR FGTCTVQKLA HQIYQFTDKD KDNVAPRSKI SPQGYG SEQ ID No: 19 - ADM-Gly 22-53 (aa 115-147 of pre-proADM SEQ ID No. 11)

[0582] 10 20 30 TVQKLAHQIY QFTDKDKDNV APRSKISPQG YG SEQ ID No: 20 - ADM-Gly 27-53 (aa 120-147 of pre-proADM SEQ ID No. 11)

[0583] 10 20 AHQIYQFTDK DKDNVAPRSK ISPQGYG SEQ ID No: 21 - ADM-Gly 33-53 (aa 126-147 of pre-proADM SEQ ID No. 11)

[0584] 10 20 FTDKDKDNVA PRSKISPQGY G SEQ ID No: 22 - bio-ADM 2-52 (aa 96-146 of pre-proADM SEQ ID No. 11)

[0585] 10 20 30 40 RQSMNNFQGL RSFGCRFGTC TVQKLAHQIY QFTDKDKDNV APRSKISPQG Y-CONH2 SEQ ID No: 23 - bio-ADM 8-52 (aa 102-146 of pre-proADM SEQ ID No. 11)

[0586] 10 20 30 40 FQGLRSFGCR FGTCTVQKLA HQIYQFTDKD KDNVAPRSKI SPQGY-CONH2 SEQ ID No: 24 - bio- ADM 22-52 (aa 115-146 of pre-proADM SEQ ID No. 11) 10 20 30 TVQKLAHQIY QFTDKDKDNV APRSKISPQG Y-CONH2

[0587] SEQ ID No: 25 - bio-ADM 27-52 (aa 120-146 of pre-proADM SEQ ID No. 11)

[0588] 10 20 AHQIYQFTDK DKDNVAPRSK ISPQGY-CONH2

[0589] SEQ ID No: 26 - bio-ADM 33-52 (aa 126-146 of pre-proADM SEQ ID No. 11)

[0590] 10 20

[0591] FTDKDKDNVA PRSKISPQGY-CONH2

[0592] SEQ ID No: 27 - (aa 143-147 of pre-proADM SEQ ID No. 11)

[0593] PQGYG SEQ ID No: 28 - (aa 143-146 of pre-proADM SEQ ID No. 11)

[0594] PQGY SEQ ID No: 29 - human serum albumin signal sequence

[0595] 10 20

[0596] MKWVTFISLL FLFSSAYSFR SEQ ID No: 30 - Prepro-PAM isoform 1 AS 1-973 (Canonical sequence of PAM)

[0597] 10 20 30 40 50 MAGRVPSLLV LLVFPSSCLA FRSPLSVFKR FKETTRPFSN ECLGTTRPW

[0598] 60 70 80 90 100 PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR IPVDEEAFVI DFKPRASMDT

[0599] 110 120 130 140 150 VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA NILYAWARNA PPTRLPKGVG

[0600] 160 170 180 190 200 FRVGGETGSK YFVLQVHYGD ISAFRDNNKD CSGVSLHLTR LPQPLIAGMY

[0601] 210 220 230 240 250 LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH VFAYRVHTHH LGKWSGYRV

[0602] 260 270 280 290 300 RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF GDLLAARCVF TGEGRTEATH

[0603] 310 320 330 340 350 IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT QNVAPDMFRT IPPEANIPIP

[0604] 360 370 380 390 400 VKSDMVMMHE HHKETEYKDK IPLLQQPKRE EEEVLDQGDF YSLLSKLLGE

[0605] 410 420 430 440 450 REDVVHVHKY NPTEKAESES DLVAEIANVV QKKDLGRSDA REGAEHERGN

[0606] 460 470 480 490 500 AILVRDRIHK FHRLVSTLRP PESRVFSLQQ PPPGEGTWEP EHTGDFHMEE

[0607] 510 520 530 540 550 ALDWPGVYLL PGQVSGVALD PKNNLVIFHR GDHVWDGNSF DSKFVYQQIG

[0608] 560 570 580 590 600 LGPIEEDTIL VIDPNNAAVL QSSGKNLFYL PHGLSIDKDG NYWVTDVALH

[0609] 610 620 630 640 650 QVFKLDPNNK EGPVLILGRS MQPGSDQNHF CQPTDVAVDP GTGAIYVSDG

[0610] 660 670 680 690 700 YCNSRIVQFS PSGKFITQWG EESSGSSPLP GQFTVPHSLA LVPLLGQLCV

[0611] 710 720 730 740 750 ADRENGRIQC FKTDTKEFVR EIKHSSFGRN VFAISYIPGL LFAVNGKPHF 760 770 780 790 800 GDQEPVQGFV MNFSNGEIID IFKPVRKHFD MPHDIVASED GTVYIGDAHT

[0612] 810 820 830 840 850 NTVWKFTLTE KLEHRSVKKA GIEVQEIKEA EAVVETKMEN KPTSSELQKM

[0613] 860 870 880 890 900 QEKQKLIKEP GSGVPVVLIT TLLVIPVVVL LAIAIFIRWK KSRAFGDSEH

[0614] 910 920 930 940 950 KLETSSGRVL GRFRGKGSGG LNLGNFFASR KGYSRKGFDR LSTEGSDQEK

[0615] 960 970

[0616] EDDGSESEEE YSAPLPALAP SSS SEQ ID NO: 31 - Prepro-PAM isoform 2 AS 1-868

[0617] 10 20 30 40 50 MAGRVPSLLV LLVFPSSCLA FRSPLSVFKR FKETTRPFSN ECLGTTRPW

[0618] 60 70 80 90 100 PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR IPVDEEAFVI DFKPRASMDT

[0619] 110 120 130 140 150 VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA NILYAWARNA PPTRLPKGVG

[0620] 160 170 180 190 200 FRVGGETGSK YFVLQVHYGD ISAFRDNNKD CSGVSLHLTR LPQPLIAGMY

[0621] 210 220 230 240 250 LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH VFAYRVHTHH LGKWSGYRV

[0622] 260 270 280 290 300 RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF GDLLAARCVF TGEGRTEATH

[0623] 310 320 330 340 350 IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT QNVAPDMFRT IPPEANIPIP

[0624] 360 370 380 390 400 VKSDMVMMHE HHKETEYKDK IPLLQQPKRE EEEVLDQDFH MEEALDWPGV

[0625] 410 420 430 440 450 YLLPGQVSGV ALDPKNNLVI FHRGDHVWDG NSFDSKFVYQ QIGLGPIEED

[0626] 460 470 480 490 500 TILVIDPNNA AVLQSSGKNL FYLPHGLSID KDGNYWVTDV ALHQVFKLDP

[0627] 510 520 530 540 550 NNKEGPVLIL GRSMQPGSDQ NHFCQPTDVA VDPGTGAIYV SDGYCNSRIV

[0628] 560 570 580 590 600 QFSPSGKFIT QWGEESSGSS PLPGQFTVPH SLALVPLLGQ LCVADRENGR

[0629] 610 620 630 640 650 IQCFKTDTKE FVREIKHSSF GRNVFAISYI PGLLFAVNGK PHFGDQEPVQ

[0630] 660 670 680 690 700 GFVMNFSNGE IIDIFKPVRK HFDMPHDIVA SEDGTVYIGD AHTNTVWKFT

[0631] 710 720 730 740 750 LTEKLEHRSV KKAGIEVQEI KEAEAWETK MENKPTSSEL QKMQEKQKLI

[0632] 760 770 780 790 800 KEPGSGVPVV LITTLLVIPV WL LAIAI FI RWKKSRAFGD SEHKLETSSG

[0633] 810 820 830 840 850 RVLGRFRGKG SGGLNLGNFF ASRKGYSRKG FDRLSTEGSD QEKEDDGSES

[0634] 860 866

[0635] EEEYSAPLPA LAPSSS SEQ ID No: 32 - Prepro-PAM isoform 3 AS 1-905 (amino acids 829-896 of SEQ ID No. 30 missing) 10 20 30 40 50 MAGRVPSLLV LLVFPSSCLA FRSPLSVFKR FKETTRPFSN ECLGTTRPW

[0636] 60 70 80 90 100 PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR IPVDEEAFVI DFKPRASMDT

[0637] 110 120 130 140 150 VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA NILYAWARNA PPTRLPKGVG

[0638] 160 170 180 190 200 FRVGGETGSK YFVLQVHYGD ISAFRDNNKD CSGVSLHLTR LPQPLIAGMY

[0639] 210 220 230 240 250 LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH VFAYRVHTHH LGKWSGYRV

[0640] 260 270 280 290 300 RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF GDLLAARCVF TGEGRTEATH

[0641] 310 320 330 340 350 IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT QNVAPDMFRT IPPEANIPIP

[0642] 360 370 380 390 400 VKSDMVMMHE HHKETEYKDK IPLLQQPKRE EEEVLDQGDF YSLLSKLLGE

[0643] 410 420 430 440 450 REDVVHVHKY NPTEKAESES DLVAEIANVV QKKDLGRSDA REGAEHERGN

[0644] 460 470 480 490 500 AILVRDRIHK FHRLVSTLRP PESRVFSLQQ PPPGEGTWEP EHTGDFHMEE

[0645] 510 520 530 540 550 ALDWPGVYLL PGQVSGVALD PKNNLVIFHR GDHVWDGNSF DSKFVYQQIG

[0646] 560 570 580 590 600 LGPIEEDTIL VIDPNNAAVL QSSGKNLFYL PHGLSIDKDG NYWVTDVALH

[0647] 610 620 630 640 650 QVFKLDPNNK EGPVLILGRS MQPGSDQNHF CQPTDVAVDP GTGAIYVSDG

[0648] 660 670 680 690 700 YCNSRIVQFS PSGKFITQWG EESSGSSPLP GQFTVPHSLA LVPLLGQLCV

[0649] 710 720 730 740 750 ADRENGRIQC FKTDTKEFVR EIKHSSFGRN VFAISYIPGL LFAVNGKPHF

[0650] 760 770 780 790 800 GDQEPVQGFV MNFSNGEIID IFKPVRKHFD MPHDIVASED GTVYIGDAHT

[0651] 810 820 830 840 850 NTVWKFTLTE KLEHRSVKKA GIEVQEIKDS EHKLETSSGR VLGRFRGKGS

[0652] 860 870 880 890 900 GGLNLGNFFA SRKGYSRKGF DRLSTEGSDQ EKEDDGSESE EEYSAPLPAL

[0653] 905

[0654] APSSS

[0655] SEQ ID No: 33 - Prepro-PAM isoform 4 AS 1-887 (amino acids 829-914 of SEQ ID No. 30 missing)

[0656] 10 20 30 40 50 MAGRVPSLLV LLVFPSSCLA FRSPLSVFKR FKETTRPFSN ECLGTTRPW

[0657] 60 70 80 90 100 PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR IPVDEEAFVI DFKPRASMDT

[0658] 110 120 130 140 150 VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA NILYAWARNA PPTRLPKGVG

[0659] 160 170 180 190 200 FRVGGETGSK YFVLQVHYGD ISAFRDNNKD CSGVSLHLTR LPQPLIAGMY

[0660] 210 220 230 240 250 LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH VFAYRVHTHH LGKWSGYRV 260 270 280 290 300 RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF GDLLAARCVF TGEGRTEATH

[0661] 310 320 330 340 350 IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT QNVAPDMFRT IPPEANIPIP

[0662] 360 370 380 390 400 VKSDMVMMHE HHKETEYKDK IPLLQQPKRE EEEVLDQGDF YSLLSKLLGE

[0663] 410 420 430 440 450 REDVVHVHKY NPTEKAESES DLVAEIANVV QKKDLGRSDA REGAEHERGN

[0664] 460 470 480 490 500 AILVRDRIHK FHRLVSTLRP PESRVFSLQQ PPPGEGTWEP EHTGDFHMEE

[0665] 510 520 530 540 550 ALDWPGVYLL PGQVSGVALD PKNNLVIFHR GDHVWDGNSF DSKFVYQQIG

[0666] 560 570 580 590 600 LGPIEEDTIL VIDPNNAAVL QSSGKNLFYL PHGLSIDKDG NYWVTDVALH

[0667] 610 620 630 640 650 QVFKLDPNNK EGPVLILGRS MQPGSDQNHF CQPTDVAVDP GTGAIYVSDG

[0668] 660 670 680 690 700 YCNSRIVQFS PSGKFITQWG EESSGSSPLP GQFTVPHSLA LVPLLGQLCV

[0669] 710 720 730 740 750 ADRENGRIQC FKTDTKEFVR EIKHSSFGRN VFAISYIPGL LFAVNGKPHF

[0670] 760 770 780 790 800 GDQEPVQGFV MNFSNGEIID IFKPVRKHFD MPHDIVASED GTVYIGDAHT

[0671] 810 820 830 840 850 NTVWKFTLTE KLEHRSVKKA GIEVQEIKGK GSGGLNLGNF FASRKGYSRK

[0672] 860 870 880 887

[0673] GFDRLSTEGS DQEKEDDGSE SEEEYSAPLP ALAPSSS SEQ ID No: 34 - Prepro-PAM Isoform 5 AS 1-974 (SEQ ID No. 30 with an additional aa in position 896)

[0674] 10 20 30 40 50 MAGRVPSLLV LLVFPSSCLA FRSPLSVFKR FKETTRPFSN ECLGTTRPW

[0675] 60 70 80 90 100 PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR IPVDEEAFVI DFKPRASMDT

[0676] 110 120 130 140 150 VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA NILYAWARNA PPTRLPKGVG

[0677] 160 170 180 190 200 FRVGGETGSK YFVLQVHYGD ISAFRDNNKD CSGVSLHLTR LPQPLIAGMY

[0678] 210 220 230 240 250 LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH VFAYRVHTHH LGKWSGYRV

[0679] 260 270 280 290 300 RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF GDLLAARCVF TGEGRTEATH

[0680] 310 320 330 340 350 IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT QNVAPDMFRT IPPEANIPIP

[0681] 360 370 380 390 400 VKSDMVMMHE HHKETEYKDK IPLLQQPKRE EEEVLDQGDF YSLLSKLLGE

[0682] 410 420 430 440 450 REDVVHVHKY NPTEKAESES DLVAEIANVV QKKDLGRSDA REGAEHERGN

[0683] 460 470 480 490 500 AILVRDRIHK FHRLVSTLRP PESRVFSLQQ PPPGEGTWEP EHTGDFHMEE

[0684] 510 520 530 540 550 ALDWPGVYLL PGQVSGVALD PKNNLVIFHR GDHVWDGNSF DSKFVYQQIG

[0685] 560 570 580 590 600 LGPIEEDTIL VIDPNNAAVL QSSGKNLFYL PHGLSIDKDG NYWVTDVALH 610 620 630 640 650 QVFKLDPNNK EGPVLILGRS MQPGSDQNHF CQPTDVAVDP GTGAIYVSDG

[0686] 660 670 680 690 700 YCNSRIVQFS PSGKFITQWG EESSGSSPLP GQFTVPHSLA LVPLLGQLCV

[0687] 710 720 730 740 750 ADRENGRIQC FKTDTKEFVR EIKHSSFGRN VFAISYIPGL LFAVNGKPHF

[0688] 760 770 780 790 800 GDQEPVQGFV MNFSNGEIID IFKPVRKHFD MPHDIVASED GTVYIGDAHT

[0689] 810 820 830 840 850 NTVWKFTLTE KLEHRSVKKA GIEVQEIKEA EAVVETKMEN KPTSSELQKM

[0690] 860 870 880 890 900 QEKQKLIKEP GSGVPVVLIT TLLVIPVVVL LAIAIFIRWK KSRAFGADSE

[0691] 910 920 930 940 950 HKLETSSGRV LGRFRGKGSG GLNLGNFFAS RKGYSRKGFD RLSTEGSDQE

[0692] 960 970

[0693] KEDDGSESEE EYSAPLPALA PSSS SEQ ID No: 35 - Prepro-PAM Isoform 6 AS 1-955 (amino acids 897-914 of SEQ ID No. 30 missing)

[0694] 10 20 30 40 50 MAGRVPSLLV LLVFPSSCLA FRSPLSVFKR FKETTRPFSN ECLGTTRPW

[0695] 60 70 80 90 100 PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR IPVDEEAFVI DFKPRASMDT

[0696] 110 120 130 140 150 VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA NILYAWARNA PPTRLPKGVG

[0697] 160 170 180 190 200 FRVGGETGSK YFVLQVHYGD ISAFRDNNKD CSGVSLHLTR LPQPLIAGMY

[0698] 210 220 230 240 250 LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH VFAYRVHTHH LGKWSGYRV

[0699] 260 270 280 290 300 RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF GDLLAARCVF TGEGRTEATH

[0700] 310 320 330 340 350 IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT QNVAPDMFRT IPPEANIPIP

[0701] 360 370 380 390 400 VKSDMVMMHE HHKETEYKDK IPLLQQPKRE EEEVLDQGDF YSLLSKLLGE

[0702] 410 420 430 440 450 REDVVHVHKY NPTEKAESES DLVAEIANVV QKKDLGRSDA REGAEHERGN

[0703] 460 470 480 490 500 AILVRDRIHK FHRLVSTLRP PESRVFSLQQ PPPGEGTWEP EHTGDFHMEE

[0704] 510 520 530 540 550 ALDWPGVYLL PGQVSGVALD PKNNLVIFHR GDHVWDGNSF DSKFVYQQIG

[0705] 560 570 580 590 600 LGPIEEDTIL VIDPNNAAVL QSSGKNLFYL PHGLSIDKDG NYWVTDVALH

[0706] 610 620 630 640 650 QVFKLDPNNK EGPVLILGRS MQPGSDQNHF CQPTDVAVDP GTGAIYVSDG

[0707] 660 670 680 690 700 YCNSRIVQFS PSGKFITQWG EESSGSSPLP GQFTVPHSLA LVPLLGQLCV

[0708] 710 720 730 740 750 ADRENGRIQC FKTDTKEFVR EIKHSSFGRN VFAISYIPGL LFAVNGKPHF

[0709] 760 770 780 790 800 GDQEPVQGFV MNFSNGEIID IFKPVRKHFD MPHDIVASED GTVYIGDAHT 810 820 830 840 850 NTVWKFTLTE KLEHRSVKKA GIEVQEIKEA EAVVETKMEN KPTSSELQKM

[0710] 860 870 880 890 900 QEKQKLIKEP GSGVPVVLIT TLLVIPVVVL LAIAIFIRWK KSRAFGGKGS

[0711] 910 920 930 940 950 GGLNLGNFFA SRKGYSRKGF DRLSTEGSDQ EKEDDGSESE EEYSAPLPAL

[0712] 955

[0713] APSSS SEQ ID No: 36 - XTEN

[0714] SESATPESGPGSEPATSGSETPGTSESATPESC

[0715] Examples

[0716] Example 1: Measurement of cerebral blood flow by transcranial contrast-enhanced ultrasound

[0717] Transcranial contrast-enhanced ultrasound (tCEU) is a non-invasive method developed and described by Premilovac et al. 2020, ensuring a standardized approach to quantitatively assess CBF changes in rodents (Premilovac et al. 2020. Journal of Cerebral Blood Flow & Metabolism. 40(5):939-953).

[0718] Briefly, the procedure involves removing fur from the animal head (rat), positioning a transducer over the head, and infusing phospholipid microbubbles to enhance ultrasound imaging. The rate of intravenous microbubble infusion was optimized at 50 ml / min to obtain a strong, but not saturated CEU signal from the brain vasculature and limit total blood volume expansion due to microbubble infusion. High-intensity ultrasound pulses are used to destroy microbubbles, and the refill of microbubbles through the cerebral vasculature is recorded for 60 seconds and quantified to calculate blood volume, flow velocity, and vascular perfusion. tCEU imaging is performed at baseline, during the MCAO, immediately after MCAO (at recanalization) and 24-hours after inducing MCAO.

[0719] Data are analyzed by fitting the acoustic intensity of microbubble refill over time to an exponential function to derive blood volume and flow velocity. Vascular perfusion is calculated as the product of these values.

[0720] Example 2: Induction of ischemic stroke via middle cerebral artery occlusion in rodent The procedure to stroke induction via middle cerebral artery occlusion (MCAO) was previously described by Sutherland BA and Buchan AM, 2013 (Sutherland and Buchan 2013. J Cereb Blood Flow Metab. 33(ll):el-7). Briefly, rats are anesthatised using isoflurane and microsurgery performed to isolate the right common carotid artery. A silicon-tipped filament is advanced through the external carotid artery up the internal carotid artery to the origin of the middle cerebral artery, where it is lodged to induce MCAO. The filament is left in place for 60 minutes before being withdrawn to allow for middle cerebral artery recanalization. After this, the wound will be sealed using nylon sutures and animals allowed to recover for 24 hours. Example 3: PEGylation of PAM

[0721] For PEGylation procedure, 10 mg of recombinant PAM (Seq ID No, 10) were diluted in PBS buffer (pH 10.0) to a concentration of 1.852 mg / mL.

[0722] Subsequently, 550 pl of NHS labelled PEG (PEG 5000, 20 mM dissolved in 20% (w / v) DMSO) were added to 5500 pl of recombinant PAM with a concentration of 1.852 mg / mL to result in a total volume of 6050 pl and an approximate 90-120 times molar excess of NHS-PEG to PAM. The NHS moiety thereby reacts with free amines on the surface of the recombinant protein. The reaction was allowed to proceed for 120 minutes at 4°C and constant agitation at 100 rpm. After 120 minutes, 500 pl of IM Tris was added to stop the reaction and the reaction was further incubated for additional 30 minutes at 4°C and constant agitation at 100 rpm. Afterwards free inactivated NHS-PEG was separated from PEGylated PAM using size exclusion chromatography (Superdex 200 16 / 600-pg, Cytiva) in combination with the Akta Start System. PEGylated PAM eluted as a defined Peak from the Column. PEG-PAM containing fractions, verified via SDS-PAGE, were pooled and subjected to the PAM- AMA Activity Assay to determine the Activity and thereby the Activity Units of the preparation (described in Example 4). The PEGylated protein was stored at -80°C until use. Figure 3 shows a typical SDS-PAGE showing PEGylated PAM (PEG-PAM) and non-PEGylated PAM (PAM).

[0723] Example 4: Determination of PAM Activity (PAM-AMA)

[0724] Amidating activity (AMA) was determined as follows: Each sample (20pl, chelator-free plasma or serum, or buffered solution containing amidating activity) was diluted two-fold in 100 mM Tris-HCl in duplicate. The amidation reaction was initiated by addition of 160 pl of PAM-reaction buffer (100 mM Tris-HCl, pH 7.5, 6.25 pM CuSCL, 2.5 mM L-ascorbate, 125 pg / mL catalase, 62.5 pM amastatin, 250 pM leupeptin, 36 ng / mL synthetic ADM-Gly (SEQ ID No.: 14)) and 375 pg / mL NT-ADM antibody (HAM1101)). Afterwards, 100 pl of each individual reaction of duplicated samples were combined and transferred into 20 pl of 200 mM EDTA to terminate the amidation reaction and to generate t=0 minutes reaction time-point followed by incubation at 37°C for 40 minutes. Afterwards the non-terminated reactions were stopped with lOpl of 200 mM EDTA. To determine the PAM activity, bio- ADM as reaction product was quantified in each sample using the sphingotest® bio- ADM immunoassay (Weber et al. 2017) as described in Example 5. The amidation assay was calibrated using a 6-point calibration curve generated with human recombinant PAM of known activity. Samples and calibrators were treated in the same manner. Relative light units (RLU t40min-t0min) determined via sphingotest® bio-ADM immunoassay for each sample were fitted against the RLU (t40min-tOmin) of the calibrator to determine the PAM activity in the samples. PAM activity is described as “amidating activity” (AMA) and is specified in Units / L, wherein 1 Unit is defined as 1 pg bio-ADM formed per hour. Units / L describe PAM activity as 1 pg bio-ADM formed per hour and L of sample.

[0725] A typical PAM calibration curve is shown in Figure (Fig.) 4A. The distribution of AMA in Li-Heparin samples from n=120 self-reported healthy volunteers are shown in Figure 4B. The median [IQR] of Li-Heparin AMA was 18.4 pg / (L*h) [13.5-21.9], The 10thand 90thpercentile was 10.5 and 24.2 pg / (L*h), respectively. The 2.5th, 97.5thand 99thpercentile was 8.1, 31.6 and 40.8 pg / (L*h). The distribution of AMA in serum was determined in 4942 samples of normal (healthy) subjects (Swedish single-center prospective population-based Study (MPP-RES)), as reported in Kaufmann et al. 2021. The median [IQR] of AMA in serum was 12.5 pg / (L*h) [10.9-14.5], The 10th and 90th percentile was 9.4 and 16.7 pg / (L*h), respectively. The 2.5th, 97.5th and 99th percentile was 7.7, 20.1 and 22.8 pg / (L*h).

[0726] Matched serum samples from n=20 subjects were measured and revealed a highly significant correlation (r = 0.89; p < 0.0001) (Figure 4C), although AMA values in serum were approximately 40% lower when compared to Li-Heparin

[0727] The distribution of PAM concentration in serum was determined in 4942 samples of normal (healthy) subjects (Swedish single-center prospective population-based Study (MPP-RES)), as reported in Ilina et al. 2023. The median [IQR] of PAM concentration in serum was 78.6 ng / mL [66.4-92.5], The 10th and 90th percentile was 56.3 and 106.6 ng / mL, respectively. The 2.5th, 97.5th and 99th percentile was 45.0, 123.5 and 135.5 ng / mL.

[0728] AMA Assay Calibration

[0729] Recombinant PAM was purchased from SinoBiological (cat-no.: 13624-H08H). A 6-point calibrator was prepared by dilution of recombinant PAM in 100 mM Tris-HCl pH 7.5, 2.5% BSA to the final concentration of 4000 ng / mL, 2000 ng / mL, 666.7 ng / mL, 222.2 ng / mL, 74 ng / mL and 0 ng / mL. Internal assay controls were undiluted and threefold diluted human serum. Amidating activity (AMA) of calibrators and controls was defined by subjecting the samples to an amidation assay: calibrators (20 pL) or controls (20 pL) were pipetted into 96 well polypropylene plates prefilled with 20 pL of 100 mM Tris-HCl, pH 7.5. Subsequently, 160 pL of reaction buffer (RB) (100 mM Tris-HCl, 6.25 μM CuSO4, 2.5 mM 1-ascorbate, 125 pg / mL bovine liver Catalase, 62.5 μM Amastatin, 125 μM Leupeptin, 375 pg / mL N-terminal anti-ADM antibody (HAM110132), 36 ng / mL synthetic human 1-53 ADM-Gly) were added. To generate a time-point 0 min, 100 pL of each individual reaction were inactivated with 20 mM EDTA. All reactions were incubated without aspiration at 37 °C for 40 min. Finally, the remaining samples were inactivated with 20 mM EDTA and subsequently subjected to the sphingotest bio-ADM assay as described in Example 6. The concentration of generated bio-ADM was used to define the AMA of each calibrator-point (formula 1).

[0730] Formula 1:

[0731]

[0732] DF: Dilution Factor of the sample

[0733] The resulting activity is measured in units / L (units per Liter of sample), where 1 Unit is defined as 1 pg bio-ADM generated within of 1 hour. The molecular weight of the bio-ADM peptide is defined as the sum of weights (in Dalton) of all Atoms in the peptide. Thereby Hydrogen has the weight of 1 Da. The molecular weight of bio-ADM is therefore 6028.9 Dalton.

[0734] Each calibrator- and control sample were analysed in duplicate in 4 independent experiments. The typical calibrator-activity was in the range of 0.7 and 122 units / L.

[0735] A person skilled in the art will understand, that due to the known concentration of PAM in the used calibrator material, it is possible to determine not only the Units of the unknown sample, but also to estimate the concentration of active PAM in the sample by fitting the determined RLU signals vs. the known calibrator concentration.

[0736] Example 5: Application of single PAM subunits for enhancing PAM activity in circulation For certain cases, increasing PAM activity via the administration of single PAM subunits, either alone or in combination, can be beneficial.

[0737] Soluble PHM and PAL subunits, comprising 31-377 residues (SEQ ID No. 9) and 495-817 residues (SEQ ID No. 8) of human PAM (UniProtKB: P19021-1), respectively, were synthesized and separately expressed in transiently transfected human kidney 293 (HEK293) cell. Both constructs were N-terminally truncated with deca-histidine tag separated by the GS linker followed by the TEV cleavage site. The signal sequences of PHM and PAL constructs were replaced with human serum albumin signal sequence (MKWVTFISLLFLFSSAYSFR [SEQ ID No. 29]). The cDNAs were cloned into an expression vector (plasmid DNA) using a 5 ’ -Notl and a 3 ’ Hindlll restriction site. The expression vectors harbouring the cDNA for PHM and PAL expression were separately replicated in- and prepared from E. coli. as a low-endotoxin preparation.

[0738] The transfection and the cultivation of the transfected HEK-INV cells with PAL or PHM constructs was performed as described for the full-length PAM in Variant A (see above).

[0739] The overexpressed recombinant constructs PHM and PAL were purified by cobalt affinity chromatography with >95% sample purity as measured by capillary gel electrophoresis. The elution fractions obtained from the protein purification process were subjected to analysis by Western blotting. Specifically, the elution fractions were loaded onto a sodium dodecyl sulphate (SDS) gel and transferred onto a nitrocellulose membrane. The membrane was probed with anti-His antibodies to detect the target protein. The fraction containing PHM or PAL proteins were pooled, dialyzed against 50 mM Tris HC1, 150 mM NaCl, pH 8.0, sterile filtered (0.2 pm). Endotoxin load was determined by Charles River PTS Endosafe system and was below 5 EU / mL.

[0740] The application of subunits, either a combination of PAL and PHM subunits (Group 1) or the PHM subunit alone (Group 2), was conducted via intravenous injection (i.v.) at a volume of 500 pL per animal in male Wistar rats (8 weeks old). The third control group received a vehicle (IxPBS). Each group consisted of six animals. Group 2 received 100 pg recombinant PHM i.v. per animal, while Group 1 received a combination of 100 pg PHM and 100 pg PAL i.v. per animal. For blood sample collection and sample application, rats were provided with a catheter in the jugular vein three days prior to blood sampling. The rats were treated twice with Carprofen (5 mg / kg), and the catheter was rinsed daily with Li-Heparin.

[0741] Fourteen sampling time points were established post-administration for each group. For each group, animals #1-3 were sampled at 0, 20 min, 60 min, 2h, and 4h, while animals #4-6 were sampled at 0, 40 min, 80 min, 3h, and 4h. The sample size was 100 pL of Li-Heparin plasma per time point. Plasma was prepared within 30 minutes after collection, shock frozen on dry ice, and stored at -80°C. The amidating activity was determined in all samples as described in Example 4. The application of the vehicle did not significantly increase PAM activity, which remained constant at an average of 6200 PAM Units throughout the experiment. For Group 2 (PHM+PAL i.v. application), the highest activity was observed at 20 minutes post-i.v., reaching nearly 45000 PAM Units, approximately a 625% elevation compared to the baseline. In Group 1 (PHM i.v. application), the highest activity was also observed at 20 minutes post-i.v., reaching nearly 20000 PAM Units, approximately a 220% elevation compared to the baseline (Fig. 5). These elevated activities remained significantly above baseline levels until approximately 1 hour before decreasing to near baseline levels by 4 hours. This data suggests that the application of PHM alone or in combination with the PAL subunit is a valid method for increasing amidating activity in circulation.

[0742] Example 6. ADM Measurements

[0743] Quantification of bio- ADM was conducted using the sphingotest bio- ADM assay as described elsewhere (Weber et al. 2017. J. Appl. Lab. Med. An AACC PubL 2, 222-233): 96-well high binding polystyrene microtiter plates (Greiner Bio-One International AG) were coated (18 h at 20 °C) with monoclonal anti-ADM antibody, directed towards amino-acids 21-32 of bio- ADM (SEQ ID No.: 15, 22-26, 28) (HAM2203, 1 pg / 0.2 mL per well in 50 mM Tris-HCl, 100 mM NaCl, pH 7.8). After blocking with 30 g / L Karion, 5 g / L BSA (protease free), 6.5 mmol / L monopotassium phosphate, 3.5 mmol / L sodium dihydrogen phosphate (pH 6.5), the plates were vacuum-dried.

[0744] 100 pL of samples / calibrators were pipetted into coated microtiter plates. Afterwards 150 pL of MACN labelled tracer antibody (HAM2302, directed towards the amidated C-terminus of bio-ADM (SEQ ID No.: 15, 22-26, 28) the microtiter plates were incubated for Ih at 22 °C under agitation at 600 rpm. Unbound tracer was removed by washing 5 times (each 350 pL per well) with washing solution (20 mM PBS, 1 g / L Triton X-100, pH 7.4). Well-bound chemiluminescence was measured for 1 s per well by using the Centro LB 960 microtiter plate luminescence reader (Berthold Technologies). The assay was calibrated using dilutions of synthetic human bio-ADM (American Peptide Company).

[0745] The lowest calibrator did not contain bio-ADM, but a concentration of 2 pg / mL was assigned to facilitate logarithmic evaluation. The calibrators were lyophilized in 20 mmol K2PO4, 6 mmol / L Na-EDTA, 5 g / L BSA, 100 pmol / L leupeptin, 50 pmol / L amastatin, 10 pg / mL of anti-N- terminal antibody HAM1101, pH 8.0, and reconstituted in H2O before use.

[0746] Example 7: Effect of PAM administration on cerebral blood flow in the cortical and striatal region following MCAO In this experiment the role of PAM administration on recovery of cerebral blood flow in ischemia-induced rats in striatum and cortex was investigated. The experiment involves the use of twenty-four (24) rats divided into three (3) groups of eight (8) rats each. Two groups of rats had microsurgery performed to receive MCAO for a period of 60min, as described in Example 2. The rats in the SHAM group underwent the microsurgery with no occlusion of the MCAO. The first MCAO group received a bolus injection of PAM (dose: 200 pg / kg body weight) via the intraperitoneal route 24h prior MCAO induction. The used PAM material was recombinant PAM (NCBI accession number NP_620176.1) that was PEGylated with PEG 5000 (polyethylene glycol with the molecular weight of 5 kDa) to extend its half-life and stability in vivo (PEG-PAM), PEGylation of PAM was performed as described in Example 3.

[0747] The second MCAO group and the sham group received a bolus injection of IxPBS via the intraperitoneal route, serving as the vehicle control. All rats in each group underwent transcranial contrast-enhanced ultrasound prior to, during, immediately after and 24 hours after MCAO induction, as described in Example 1. For analysis of CEU recordings, all data was analysed using QLab advanced quantification software (Phillips). Regions of interest were drawn in the motor / sensory cortical regions and the striatal / thalamus brain regions. These regions were selected carefully to exclude contributions of large arteries where possible as these can dominate the overall result due to signal intensity.

[0748] As shown in the Fig. 6A and Fig. 6C all animals as expected had a similar cerebral blood flow in cortex and striatum, respectively, measured in Al / sec, in all groups at baseline. After induction of MCAO, the MCAO + Vehicle and MCAO + PEG-PAM groups show the expected decline to 11.6 and 5.9 % in cerebral blood flow in cortex region, respectively, compared with the respective cerebral blood flow level at the baseline (Fig. 6B), while the sham animals retain a stable cerebral blood flow. 24h post MCAO, the cerebral blood flow in the sham + Vehicle group remained stable and. Surprisingly, in the MCAO + PEG-PAM group cerebral blood flow has returned to baseline levels, showing almost 90% recovery, in the cortical regional while the MCAO + Vehicle group was only 33% of the baseline pre-MCAO level (Fig.6B). Similarly to cortex, in striatal region after induction of MCAO, the MCAO + Vehicle and MCAO + PEG-PAM groups show the expected decline to 5.7 % and 6.6 % in cerebral blood flow, respectively, compared with the respective cerebral blood flow level at the baseline (Fig. 6D). 24h post MCAO, the cerebral blood flow in striatal region in the MCAO + PEG-PAM group was recovered to 65.4 % when compared to its pre-MCAO baseline level, while the recovery in the MCAO + Vehicle group was only 41.2 % when compared to the baseline pre-MCAO level (Fig. 6D). These data suggest that PAM, especially long-lasting PEG-PAM is a potent agent for increasing cortical and striatal hemodynamics, such as cerebral blood flow, in ischemia.

[0749] Example 8: Effect of PAM administration on capillary function and integrity in the cortical region following MCAO

[0750] Cerebral blood flow and the capillary function are closely linked together. Thus, along with other known mechanisms, CBF can be controlled by capillary density, capillary recruitment, and permeability of BBB (reviewed in Andjelkovic et al. 2020. Fluids Barriers CNS 17, 44 and Yoshiaki et al. 2012. Journal of Cerebral Blood Flow & Metabolism 32, 1167-1176). Under normal conditions, endothelial cells, being one of the main components of the blood-brain barrier and lacking a contractile apparatus, play a pivotal role in controlling cerebral blood flow by releasing vasoactive mediators to smooth muscle cells (SMCs). The disturbance of endothelial cells under pathological conditions results in vasogenic edema and is known to compress capillaries, disturbing capillary flow when brain edema increases intracranial pressure. The endothelial cells themselves become swollen under ischemia and may impede capillary flow in the brain after ischemia / reperfusion, causing a 'no-reflow' phenomenon (Chians et al. 1968. Am J Pathol 52:455-76). Damage to the endothelial cells can expose subendothelial connective tissue, prompting platelet thrombosis and disturbing capillary flow. Upon changes in their chemical microenvironment, endothelial cells on the capillaries may express many types of adhesion molecules and trap various leukocytes (Lindauer et al. 1996. J Cereb Blood Flow Metab 16: 1143-52). Adhering leukocytes may plug the capillaries, disturbing capillary flow (del Zoppo et al. 1991. Stroke 22:1276-83). Additionally, capillary density, e.g., through angiogenesis, and capillary recruitment affect the CBF by increasing the surface area available for blood flow and also support tissue survival in the brain tissue by providing selected nutrients. Studies have shown that in stroke, increased angiogenesis, which is associated with enhanced capillary density, significantly improves CBF (Zhang et al. 2013. J NeuroEngineering Rehabil 10, 43).

[0751] In this experiment the role of PAM administration on vascular integrity, specifically capillary function, in ischemia-induced rats in cortex was investigated. The experimental design, including the number of rats, grouping, and treatment protocol, is described in Example 7. The capillary function was accessed using ex vivo fluorescent vascular cast technique and laminin labeling. Briefly, after the 24-hour recovery period post-MCAO, rats were overdosed with pentobarbitone sodium (200mg / kg) and transcardially perfused using PBS, followed by 4% paraformaldehyde, followed by 5% gelatin containing 0.2% FITC-dextran (lOkD) to create an in-situ fluorescent vascular cast. The pressure during each step of the perfusion was monitored using an inline sphygmomanometer and maintained at -lOOmmHg. Brains were then be excised and processed for cryo-sectioning.

[0752] A first series of brain sections (coronal brain sections (40pm)) were collected and mounted onto IHC Flex slides and stored at -20°C. A series of brain sections (1 section per mm brain) from each animal was used to determine infarct volume using cresyl violet staining as well as laminin staining on the same slide. Laminin expression is known to increase in the vascular basement membrane following ischemic injury, and the extent of laminin staining has been shown to correlate with total infarct volume.

[0753] Therefore, laminin serves as a reliable biomarker for stroke severity. In this study, the ratio of vascular ischemic volume to total infarct volume was calculated and compared between the MCAO + -PEG-PAM and MCAO + vehicle groups.

[0754] A second series of brain sections underwent immunohistochemistry to label all blood vessels basement membranes (Lycopersicon Esculentum [Tomato] Lectin-647) and 4',6-diamidino-2-phenylindole (DAPI) was used to label cell nuclei. Capillary perfusion in the cortical region affected by MCAO was quantified by measuring the fluorescent signal. The signal in the sham + vehicle group was set to 100% and used as a reference for comparison with the two MCAO groups. A higher fluorescent signal indicates healthy, intact capillaries.

[0755] All brain sections were imaged using the VS 120 Slide Scanner. Image analysis was undertaken using ImageJ and QuPath.

[0756] As shown in Fig. 7 A, capillary perfusion in the cortex decreased by approximately 36% in the MCAO + vehicle group compared to the sham + vehicle group. In contrast, the MCAO + PEG-PAM group exhibited a significantly smaller reduction, with capillary perfusion decreasing by only 15%. Additionally, animals in the MCAO + PEG-PAM group showed a reduction in the laminin-to-infarct volume ratio (to 0.8), compared to the ratio of 1.1 observed in vehicle-treated animals (Fig. 7B). Collectively, these data suggest that PAM, especially long-lasting PEG-PAM exerts a protective effect on brain blood vessels in both infarct-affected and adjacent brain areas following ischemia.

[0757] Example 9: Effect of PAM administration on reduction of astrogliosis in various brain regions

[0758] Both in vivo and in vitro studies support the role of various parenchymal components, such as astrocytes, in the regulation of cerebral blood flow at the level of the arteries, arterioles, and capillaries. Astrocytes actively regulate CBF through neurovascular coupling by responding to neuronal activity, including activation of metabotropic glutamate receptors. This activation triggers calcium signaling in astrocytes, which can lead to either vasodilation or vasoconstriction of blood vessels, thereby affecting cerebral blood volume and flow. Additionally, astrocytes regulate vascular tone through arachidonic acid pathways and by influencing smooth muscle potassium channels. They also interact with non-glutamatergic neurotransmitters such as nitric oxide and vasoactive intestinal peptide, which can modulate neurovascular signaling. Astrocytes contribute to the structural integrity and function of the blood-brain barrier and participate in angiogenesis. They communicate with endothelial cells to maintain endothelial tight junctions and influence transport across the BBB (reviewed in Koehler RC, Roman RJ, Harder DR. Astrocytes and the regulation of cerebral blood flow. Trends Neurosci. 2009 Mar; 32(3): 160-9).

[0759] Under pathological conditions, including various neurological and / or vascular diseases or in states of inflammation, astrocytes undergo morphological and functional changes characterized by cell hypertrophy and the excessive release of neurotoxic factors, referred to as reactive astrocytes or astrogliosis. These changes impair astrocytic regulation of cerebral blood flow via neurovascular coupling and other mechanisms, which in turn exacerbate or contribute to disease progression.

[0760] Some key molecular biological hallmarks of astrogliosis include the upregulation of Glial Fibrillary Acidic Protein (GFAP), increased expression of pro-inflammatory cytokines and chemokines (such as interleukin- ip and tumor necrosis factor-a), changes in calcium signaling, and activation of signal transduction pathways by STAT3, NF-kB, and MAPK, which regulate the expression of genes involved in inflammation, scar formation, and cellular proliferation (reviewed in Matusova Z, Hol EM, Pekny M, Kubista M and Valihrach L (2023) Reactive astrogliosis in the era of single-cell transcriptomics. Front. Cell. Neurosci. 17: 1173200; reviewed in Lawrence, J. M., Schardien, K, Wigdahl, B. et al. Roles of neuropathology-associated reactive astrocytes: a systematic review, acta neuropathol commun 11, 42 (2023)). Along with indications that reactive astrocytes might have a neuroprotective role, several studies have shown their neurotoxic effects.

[0761] In neurodegenerative diseases such as Alzheimer’s disease, reactive astrocytes are found in proximity to amyloid-beta (AP) plaques and are implicated in the propagation of inflammatory responses (Kato S, Gondo T, Hoshii Y, Takahashi M, Yamada M, Ishihara T. Confocal observation of senile plaques in Alzheimers’ disease: senile plaque morphology and relationship between senile plaques and astrocytes. Pathol Int. 1998;48(5): 332-40). Excessive production of neurotoxic factors, such as TNF-a and IFN-y, modulates astrocyte APP processing homeostasis and tau phosphorylation, which leads to increased Ap load and toxicity (Garwood CJ, Pooler AM, Atherton J, Hanger DP, Noble W. Astrocytes are important mediators of Af-induced neurotoxicity and tau phosphorylation in primary culture. Cell Death Dis.

[0762] 2011;2(6): e!67). The APPswePSldE9 mouse model displays extensive amyloid-induced reactive astrogliosis. Thus, reducing astrogliosis has been suggested as a beneficial strategy for mitigating various neurodegenerative pathologies, including AD, which could also potentially increase cerebral blood flow (Smit T, Deshayes NAC, Borchelt DR, Kamphuis W, Middeldorp J, Hol EM. Reactive astrocytes as treatment targets in Alzheimer's disease-Systematic review of studies using the APPswePSldE9 mouse model. Glia. 2021;69(8): 1852-1881; Korte N, Nortley R, Attwell D. Cerebral blood flow decrease as an early pathological mechanism in Alzheimer's disease. Acta Neuropathol. 2020 Dec; 140(6):793-810).

[0763] In the present invention, the effect of PAM administration on the reduction of reactive astrocytes in vivo in rats with induced chronic neuroinflammation across various brain regions was investigated.

[0764] To this end, an in vivo model was employed to mimic chronic neuroinflammation, validated for memory deficits, synaptic loss, and altered apoptosis regulation, leading to an Alzheimer-like phenotype. The reactive astrocytes, as biomarker for neuroinflammation, were measured ex vivo by means of immunohistochemistry. Neuroinflammation was induced via intracerebroventricular (ICV) injection. On day 0, mice were anesthetized using a ketamine-xylazine mixture (100 and 10 mg / kg, respectively) and placed in a stereotaxic frame. After aseptic preparation of the surgical site, a 2 pL injection of amyloid-betal-42 oligomers, sourced from BACHEM, was delivered to the right lateral ventricle using a microinjection system at a rate of 1 pL / min.

[0765] Twelve 18-month-old male C57B1 / 6J mice were divided into two groups, receiving either a treatment of PEGylated recombinant PAM (200 pg / kg) or a control solution of 0.9% NaCl (10 mL / kg). The treatment regimen included doses administered intraperitoneally on a schedule starting 10 and 5 days prior to the induction of neuroinflammation and continuing on Days 0 (immediately after ICV injection), 5, 10, and 15 post-ICV. The used PAM material was recombinant PAM (NCBI accession number NP 620176.1) that was PEGylated with PEG 5000 (polyethylene glycol with the molecular weight of 5 kDa) to extend its half-life and stability in vivo (PEG-PAM), PEGylation of PAM was performed as described in Example 3. Post- operation, recovery was aided by maintaining body temperature using a feedback-controlled heating pad, and pain was managed with a post-operative subcutaneous injection of buprenorphine (0.05 mg / kg).

[0766] Brain tissue was harvested 17 days post-ICV. Mice were perfused transcardially with heparinized saline followed by 4% paraformaldehyde. Brain regions (prefrontal cortex, hippocampus, and cortex) were dissected, flash-frozen, and stored at -80°C. Cryosections (14 pm) were stained for reactive astrocytes using anti-GFAP antibodies and analyzed for fluorescence intensity using a digital imaging system. In short, the sections were acclimated to RT before proceeding with staining protocols. To reduce non-specific antibody binding, sections were immersed in a blocking solution consisting of 10% normal donkey serum and 1% bovine serum albumin in IX PBS for 90 minutes at room temperature. Following blocking, sections were incubated with a primary antibody solution containing rabbit anti-GFAP at a dilution of 1:200 in the blocking solution overnight at 4°C. The next day, sections were washed three times with PBS and subsequently incubated with a secondary antibody, donkey anti-rabbit IgG H& L conjugated to Alexa Fluor® 647, diluted 1:200 in the blocking solution. This incubation was carried out for two hours at RT, followed by the washing with PBS. Nuclear staining was performed by applying DAPI (4',6-diamidino-2-phenylindole) at a concentration of 1:5000 for 10 minutes.

[0767] Images were captured in cortex and 5 different regions of the hippocampus per hemisphere (total 12 images per animal): namely the Dentate Gyrus (DG), the three Ammon’s Hom areas (Cornu Ammonis 1 (CA1), CA2 & CA3) and the Stratum Lacunosum-Moleculare (SLM). Images were then analysed by ImageJ® software (NIH, USA) and a count of the labelled positive cells in the different target areas was carried out and expressed in cells / mm2. Comparative analysis between groups was carried out.

[0768] Fig. 8 details the specific responses of reactive astrocytes to each treatment. In regions such as the dentate gyrus (Fig. 8A), SLM (Fig. 8B), CA1 (Fig. 8C), CA2 (Fig. 8D), CA3 (Fig. 8E) and total hippocampus (Fig. 8F) the PAM treated animals surprisingly exhibited a significantly lower proportion of reactive astrocytes (measured per / mm2 or as % to the total amount of astrocytes) compared to the control (PBS-treated) group. The mean values are summarized in Tab. 1. This data suggest that PAM is a potent agent for reducing the astrogliosis in neuroinflammation. Table 1: Quantitative Comparison of Reactive Astrocytes in Various Brain Regions: Density (per mm2) and Proportion (%) in PAM-Treated vs. PBS-Treated Animals

[0769]

[0770] Example 10: Modification of PAM: Pegylation with PEG-10000, PEG-5000 via Cysteines and modification with an XTEN peptide

[0771] The modification protocols described below provide a reproducible and efficient method for modification of recombinant PAM (example 3) using Mal-PEG-5000 (5 kDa PEG), NHS-PEG-10000 (10 kDa PEG) and an XTEN peptide (SEQ ID No. 36).

[0772] The protocol: 1.5 mg of recombinant PAM in a total volume of 1.5 mL of phosphate-buff ered saline (PBS) at pH 10 was used. 1 M NaOH was added to adjust the pH to 8.5 and the preparation was divided into three equal portions, 500 pl each.

[0773] For pegylation with NHS-PEG-10000 (11153 Da, Iris Biotech GmbH, Germany), a 10 mM solution of NHS-PEG-10000 was prepared by dissolving 62 mg of PEG-10000 in 556 pL of 20% DMSO. This solution was added to the PAM solution at a molar excess of approximately 90-120-fold compared to PAM, or a 2.2-fold molar excess compared to the lysine residues present in the PAM construct. The PAM-PEG mixture was incubated for 120 minutes at 4°C with constant shaking. After 120 minutes, 50 pL of 2 M Tris solution was added to the mixture to inactivate any unreacted NHS-PEG, and incubated for an additional 30 minutes. To separate PEG-PAM from free PEG, the mixture was fractionated on the Superdex200 column (ÄKTA Start) with PBS as the running buffer. The protein peak was detected at 280 nm, and fractions corresponding to a molecular weight of 160 to 90 kDa were collected and pooled. The pooled fractions were sterile filtered with a 0.2 pM filter and aliquoted under sterile conditions.

[0774] For pegylation with MAL-PEG-5000 (4908 Da, Iris Biotech GmbH, Germany), a 1M solution of DTT was prepared and 5µl of the solution were added to the second 500 µl portion of PAM. After an incubation time of Ih at ambient temperature, the PAM protein underwent desalting using a G5 desalting column (EMP Biotech, Germany) resulting in a final volume of 1 mL. A 20 mM solution of MAL-PEG-5000 was prepared by dissolving 65 mg of MAL-PEG in 662,2 µl of 20% DMSO. This solution was added to the PAM solution at a molar excess of approximately 90-120-fold compared to PAM. After an incubation for 120 minutes at ambient temperature under constant shaking and protection from light, PEG-PAM was separated from free PEG by fractionation on the Superdex200 column (ÄKTA Start) with PBS as the running buffer. The protein peak was detected at 280 nm, and fractions corresponding to a molecular weight of 160 to 90 kDa were collected and pooled. The pooled fractions were sterile filtered with a 0.2 pM filter and aliquoted under sterile conditions.

[0775] For XTENylation with an XTEN peptide having the following amino-acid sequence: H-SESATPESGPGSEPATSGSETPGTSESATPESC-OH (SEQ ID No. 36) (3623,7 Da, custom synthesis by Peptides& Elephants GmbH, Germany), the third 500 µl portion of PAM was used. In the first step PAM was coupled to the linker MAL-beta-Ala-Osu (Maleimidobutyryloxysuccinimidylester, Iris Biotech GmbH, Germany) by adding of 15 µl of a 10 mM MAL-beta-Ala-Osu (in 20% DMSO) solution to the PAM protein to couple the linker via its N-succinimidylester to free lysine residues of the PAM protein. The reaction was incubated for 120 minutes at 4°C with constant shaking. After 120 minutes, 50 pL of 2 M Tris solution was added to the mixture to inactivate any unreacted linker and incubated for additional 30 minutes.

[0776] Afterwards the PAM protein underwent desalting into PBS pH 10 using a G5 desalting column (EMP Biotech, Germany) to remove the unreacted linker resulting in a final volume of 1 mL. A 19.3 mM solution of the XTEN-peptide was prepared in PBS pH 10 and a 100-fold molar excess of the XTEN-peptide was added to the PAM-Linker complex to couple the free C-terminal Cysteine residue of the XTEN-peptide to the maleimide moiety of the linker. After an incubation of 120 minutes at ambient temperature under constant shaking and protection from light, XTENylated PAM (X-PAM) was separated from free XTEN by fractionation on the Superdex200 column (ÄKTA Start) with PBS as the running buffer. The protein peak was detected at 280 nm, and fractions corresponding to a molecular weight of 160 to 90 kDa were collected and pooled. The pooled fractions were sterile filtered with a 0.2 pM filter and aliquoted under sterile conditions.

[0777] The protein concentration of all three PAM preparations was determined using the bicinchoninic acid kit (Micro BCA™ Protein- Assay -Kit, ThermoFisher Scientific, Cat# 23235) for protein determination.

[0778] Results: the resulting protein preparations (PEG-PAM 5000 (PEGylation via Cysteines), PEG-PAM 10000 (PEGylation via Lysines) and X-PAM (XTENylation via Lysines) were analysed by SDS-PAGE, showing a shift towards a higher molecular weight in all three preparations, when compared to untreated PAM (figure 9, right). Moreover, the enzyme activity of all preparations was determined as described in example 4 and compared to that of untreated PAM (figure 9, left). The PEGylation via Lysines using PEG- 10000 retains 88% of specific PAM activity when compared to the untreated enzyme. XTENylation via Lysines using a linker retains 76% of specific PAM activity when compared to untreated PAM, indicating that similar to PEGylation with PEG 5000 (example 3) the PEGylation with PEG 10000 does not substantially interfere with PAM activity. XTENylation resulted in a decline of specific PAM activity by 24% when compared to untreated PAM, however, this reduction may be in part caused by overestimation of total PAM concentration in the used total protein determination assay (Micro BCA™ Protein-Assay-Kit), which may be false-positively impacted by the attached XTEN peptide, leading to an underestimation of specific PAM activity. PEGylation with PEG-5000 via Cysteine residues surprisingly resulted in significant drop of specific PAM activity, indicating that this modification method is not suitable for the enzyme PAM.

[0779] In summary, the results, as illustrated in Figure 9, revealed that the activity of pegylated PAM with PEG 10000 via coupling to free Lysine residues was slightly reduced compared to nonmodified PAM. The pegylated PAM exhibited a decrease in activity by 12%. Similarly, coupling of the XTEN peptide to free Lysine residues exhibited a decrease in activity by 24%. Lastly and surprisingly, in contrast to coupling of PEG 5000 to free lysine residues, as shown in example 3, coupling of PEG 5000 to cysteines results in a substantial drop of specific PAM activity by 87%.

Claims

1. Claims1. Modified or unmodified Peptidylglycine a-amidating monooxygenase (PAM) or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of cerebral blood flow (CBF).

2. Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF according to claim 1, wherein said subject has a reduction in CBF.

3. Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of cerebral blood flow according to claim 1 or 2, wherein said subject is suffering from or has a risk of suffering from a disease or medical condition selected from the group comprising a. neuroinflammatory and neurodegenerative diseases; particularly dementia selected from the group comprising mild cognitive impairment (MCI), Alzheimer’s disease, vascular dementia, mixed Alzheimer’s disease, and vascular dementia, Lewy body dementia, frontotemporal dementia, focal dementias (including progressive aphasia), subcortical dementias (including Parkinson’s disease), secondary causes of dementia syndrome (including intracranial lesions, multiple sclerosis, Huntington’s Disease, Amyloid Lateral Sclerosis, Traumatic Brain Injury, Creutzfeldt-Jakob Disease);5.b. cardiovascular diseases or conditions, particularly selected from the group comprising atherosclerosis, hypertension, heart failure (including acute and acute decompensated heart failure), atrial fibrillation, cardiovascular ischemia, cerebral ischemic injury, cardiogenic shock, carotid artery stenosis, hypotension, hypertension, peripheral artery disease, coronary artery disease, stroke (including ischemic and haemorrhagic stroke and transient ischemic attack) and myocardial infarction;6.c. metabolic disorders, particularly diabetes mellitus type 1, diabetes mellitus type 2, obesity, and metabolic syndrome;7.d. infections caused by infectious organisms such as bacteria, viruses, fungi or parasites, particularly selected from the group comprising SIRS, sepsis, and septic shock; e. respiratory disorders, particularly (chronic obstructive pulmonary disease, sleep apnea)8.f. psychiatric conditions (major depressive disorder, bipolar disorder).

4. Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF according to claims 1 to 3, wherein said CBF is assessed with a method selected from the group comprising ultrasound-based techniques for CBF measurements (Transcranial Doppler Ultrasound (TCD); Contrast-Enhanced Ultrasound (CEU), Functional Ultrasound (fUS)), nuclear medicine methods (Single Photon Emission Computed Tomography (SPECT), Positron Emission Tomography (PET)), magnetic resonance imaging (MRI) techniques (Arterial Spin Labeling (ASL), Dynamic Susceptibility Contrast (DSC) MRI, Dynamic Contrast-Enhanced MRI (DCE-MRI), Blood Oxygenation Level-Dependent (BOLD) Functional MRI (fMRI)) and X-ray computed tomography methods (X-Ray Computed Tomography (CT) Perfusion, Xenon-Enhanced CT (Xe-CT)).

5. Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF, according to claims 1 to 4, wherein the CBF is defined as volume of blood expressed in milliliter that passes through 100 g of brain tissue per minute.

6. Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF, according to claims 1 to 5, wherein the cerebral blood flow in said subject is below a certain threshold.

7. Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF according to claims 1 to 6, wherein said PAM or fragments thereof are modified by a. attaching one or more natural or synthetic polymers units, wherein said polymers are selected from the group comprising13.i. PEG, particularly having an average molecular weight in the range of 0.2-100 kDa, particularly 1-90 kDa, more particularly 2-80 kDa, more particularly 3-70 kDa, more particularly 4-60 kDa and even more particularly 5 to 50 kDa; ii. XTEN;14.iii. a peptide polymer consisting of repeats of PAS (the amino acids proline, alanine and serine, particularly comprising 100-200, particularly 120-180, more particularly 130-170, even more particularly about 150 PAS repeats; iv. elastin-like polypeptides consisting of Valin-Prolin-Glycine-x-Glycine repeats, wherein x is any amino acid other than Proline, particularly consisting of from 150, 200, 250, 300, 400, or 500 to 550, 600, 750, 850, 950 or 1,000 amino acids; v. HAP polypeptide, having the sequence (Gly4Ser)n, wherein n is 100-200; vi. gelatin-like fusion protein, having the sequence (Gly-X-Y)n, wherein X and Y individually are any amino acid except for Cysteine, and wherein n = 60 to 1500; vii. polysaccharides, in particular those chosen from the group comprising:

1. dextrans;2. hydroxyethyls;3. heparosan;4. hyaluronic acid;19.viii. polysialic acid.20.b. conjugation with a serum protein, such as albumin or an immunoglobulin or parts of an immunoglobulin;21.c. site-directed mutagenesis by insertion, deletion and / or exchange of one or more amino acids within the amino acid sequence of PAM or fragments thereof.

8. Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF according to claim 1 to 7, wherein said PAM or fragment thereof is administered in combination with ascorbate and / or copper and / or peptide with C- terminal glycine amino acid or fragments thereof.

9. Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF.

10. Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF according to claim 9, wherein said PAM or a fragment thereof is modified by24.a. attaching one or more natural or synthetic polymers units, wherein said polymers are selected from the group comprising25.i. PEG, particularly having an average molecular weight in the range of 0.2-100 kDa, particularly 1-90 kDa, more particularly 2-80 kDa, more particularly 3-70 kDa, more particularly 4-60 kDa and even more particularly 5 to 50 kDa; ii. XTEN;26.iii. a peptide polymer consisting of repeats of PAS (the amino acids proline, alanine and serine, particularly comprising 100-200, particularly 120-180, more particularly 130-170, even more particularly about 150 PAS repeats; iv. elastin-like polypeptides consisting of Valin-Prolin-Glycine-x-Glycine repeats, wherein x is any amino acid other than Proline, particularly consisting of from 150, 200, 250, 300, 400, or 500 to 550, 600, 750, 850, 950 or 1,000 amino acids; v. HAP polypeptide, having the sequence (Gly4Ser)n, wherein n is 100-200; vi. gelatin-like fusion protein, having the sequence (Gly-X-Y)n, wherein X and Y individually are any amino acid except for Cysteine, and wherein n = 60 to 1500; vii. polysaccharides, in particular those chosen from the group comprising:

1. dextrans;2. hydroxyethyls;3. heparosan;4. hyaluronic acid;31.viii. polysialic acid.32.b. conjugation with a serum protein, such as albumin or an immunoglobulin or parts of an immunoglobulin;33.c. site-directed mutagenesis by insertion, deletion and / or exchange of one or more amino acids within the amino acid sequence of PAM.

11. Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF according to claims 9 or 10, wherein said pharmaceutical formulation is to be administered, epicutaneously, subcutaneously,intradermally, intramuscularly, intravascular (intraarterially, intravenously), or via the central nervous system (CNS, intracerebrally, intracerebroventricularly, intrathecally) or via intraperitoneal administration.

12. Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF according to claims 9 to 11, wherein said pharmaceutical formulation is a solution, preferably a ready -to-use solution.

13. Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF according to claims 9 to 12, wherein said pharmaceutical formulation is in a freeze-dried state.

14. Pharmaceutical formulation modified or unmodified comprising PAM or fragments thereof for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF according to claims 9 to 13, the formulation comprising said PAM in combination with ascorbate and / or copper and / or peptide with C-terminal Glycine amino acid or fragments thereof.

15. A kit comprising the pharmaceutical formulation according to claims 9 to 14 for use in the treatment or prevention of cerebral hypoperfusion in a subject in need thereof for the improvement of CBF.

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