Use of inactive adrenomedullin precursor for therapeutic purposes

Abstract

Subject matter of the present invention is a pharmaceutical formulation of ADM-Gly and fragments thereof in modified or unmodified form. The invention further relates to the techniques for use in a method for the treatment and / or prevention of diseases with compromised endothelial barrier, especially of cardiovascular, neurodegenerative, edematous and / or inflammatory disorders.

Description

[0001] USE OF INACTIVE ADRENOMEDULLIN PRECURSOR FOR THERAPEUTIC

[0002] PURPOSES

[0003] Subject matter of the present invention is a pharmaceutical formulation of ADM-Gly and fragments thereof in modified or unmodified form. The invention further relates to the techniques for use in a method for the treatment and / or prevention of diseases with compromised endothelial barrier, especially of cardiovascular, neurodegenerative, edematous and / or inflammatory disorders.

[0004] The present invention relates to techniques in order to achieve an elevation of bioactive adrenomedullin (bio-ADM) in vivo using non-modified wildtype biosynthetic precursor of adrenomedullin (ADM-Gly). The invention further relates to the techniques for use in a method for the treatment and / or prevention of diseases with chronic or acute endothelial dysfunction, especially of cardiovascular, neurodegenerative, edematous and / or inflammatory disorders.

[0005] State of the Art

[0006] Adrenomedullin (ADM) is one of the best studied peptide hormones, which plays a role in a vast range of physiological and pathophysiological processes, including inter alia vasodilation, angiogenesis and hormone regulation. ADM is also involved in bronchodilatation, renal function, cell growth, differentiation, neurotransmission, and modulation of the immune response. ADM belonging to the ADM / calcitonin gene-related peptide (CGRP) superfamily of peptides, and is known to be produced in various human organs and tissues, including the heart, adrenal endothelial cells, lungs, kidneys, adipose tissue, and vascular endothelium, which contribute to ADM blood levels.

[0007] ADM mRNA encodes a preprohormone of 185 amino acids (SEQ ID No. 1), the pre-pro- Adrenomedullin that is enzymatically converted into Proadrenomedullin by cleavage of the N-terminal signal peptide. Proadrenomedullin is then further process by several prohormone convertases to result in four peptides, namely

[0008] PAMP (SEQ ID No. 2): Proadrenomedullin amino-terminal peptide or Proadrenomedullin N- terminal 20 peptide with a C-terminal glycine residue,

[0009] MR-proADM (SEQ ID No. 3): Mid-regional Proadrenomedullin, a stable and inert peptide, ADM-Gly (SEQ ID No. 4): C-terminally glycine extended, inactive precursor of biologically active ADM (bio-ADM (SEQ ID No. 5) and

[0010] CT-proADM (SEQ ID No. 6): C-terminal Proadrenomedullin or Adrenotensin.

[0011] ADM-Gly is the direct, inactive biosynthetic precursor of the fully activated ADM form (bio-ADM), often referred to as the intermediate form of ADM, and represents the dominating circulating form of ADM in humans. To gain its biological activity, ADM-Gly is activated by the Vitamin C dependent enzyme peptidylglycine-alpha amidating monooxygenase (PAM). The ADM processing pathway is shown in Fig. 1. PAM recognizes the C-terminal glycine and catalyses a sequential two-step reaction also referred to as amidation or C-terminal amidation. Thereby glyoxylate, originating from the C-terminal glycine, is released and an amidated carboxyl terminal tyrosine structure in ADM-Gly is formed.

[0012] Little is known about behaviour of ADM-Gly or it’s levels in clinical settings, but the analyses of bio- ADM as a target and drug-candidate in therapy was investigated in several preclinical and clinical studies. It is important to note, that term “Adrenomedullin” (ADM), as used in many publications is imprecise and in terms of this invention it is to distinguish between fully active bio-ADM and it is direct inactive C-terminally glycine extended precursor ADM-Gly (summarized in Eggelkraut-Gottanka, R. v., Curr. Med. Chem. 11, 2651 2665 (2004)).

[0013] In heart-failure, administration of bio-ADM reduced the size of the necrosis zone in myocardial infarction, apoptosis of cardiac myocytes, pronounced remodeling of the left ventricle (in animals), and aldosterone levels (in animals and humans); improvement in hemodynamic parameters (in both humans and animals) and survival (in animals) (see e.g. Niu, P. et al. Hypertens. Res. 26, 731 736 (2003), and Balint, L et al., Circulation Research, 132(9), 1185-1202, 2023.) Continuous administration of bio- ADM in 7 patients with acute heart failure led to a decrease in mean arterial pressure (MAP), pulmonary artery pressure, systemic and pulmonary vascular resistance, and also led to an increase in cardiac output. In patients with congestive heart failure (HF), infused bio-ADM lowered mean arterial pressure and increased heart rate, but to a lower extent when compared to healthy individuals. Bio-ADM infusion lowered the pulmonary arterial pressure in HF patients, but not in healthy patients. Further, increase in cardiac index was observed, an increase in urine volume and sodium excretion and a decrease in aldosterone levels, both in healthy in patients with HF (reviewed in: Balint, L. et al, Circulation Research, 132(9), 1185-1202, 2023).

[0014] Further, bio-ADM has several physiological effects, such as vasodilation, angiogenesis, cardioprotection, nephroprotection, anti-oxidation, anti-apoptosis and tissue repair and regeneration. Bio-ADM is involved in blood pressure regulation, bronchodilatation, renal function, hormone secretion, cell growth, differentiation, neurotransmission, and modulation of the immune response. Moreover, ADM plays a crucial role as autocrine factor during proliferation and regeneration of endothelial cells.

[0015] Additionally, bio-ADM promotes angiogenesis, arteriogenesis, prevents cognitive decline after chronic cerebral hypoperfusion and is therefore considered as therapeutic agent in vascular dementia (reviewed in Garcia, M. A. et al. Expert Opin. Ther. Targets 10, 303 317 (2006) and Balint, L., et al.Circulation Research, 132(9), 1185-1202, 2023).

[0016] In infectious diseases, such as sepsis, severe pneumonia or septic shock, bio-ADM concentrations in the blood-stream increase markedly (summarized in Kita, T. et al., Hypertens. Res. 45, 389 400 (2022)). Several reports in literature show that ADM is indispensable for an integral endothelial barrier function (Van Lier, et al., J. Intern. Med. 289, 792 806 (2021)). Administration of ADM to supra-physiological levels exerts strong anti-edematous and anti-inflammatory functions in a variety of inflammatory conditions in animal experiments including sepsis, acute lung injury and inflammation of the intestine (reviewed in Temmesfeld-wollbruck, B. et al., 944 951 (2007)). Further, elevated circulating levels of bio-ADM were observed in- and associated with hypertension, renal failure, inflammatory bowel disease (IBD) and pancreatitis (Ashizuka, S. et al., Biomedicines 9, (2021)). Despite the elevation of bio-ADM in clinical manifestations of diseases, such as IBD, therapeutic administration of bio-ADM showed positive effects on disease outcome (Eto, T. et al., Nephron 89, 121 134 (2001), Ueda, S. et al., Am. J. Respir. Crit. Care Med. 160, 132 136 (1999). Additionally, high levels of pro-Adrenomedullin or a fragment thereof (which is not mature ADM-NHj) in the circulation seem to indicate the need of the body to repair the function of the vascular endothelium and the need to support vascular integrity. However, low levels of mature ADM (mature ADM-NHj) indicate, that despite of high levels of pro- ADM the conversion from ADM-Gly to mature ADM (mature ADM-NHj) seems to be disturbed.

[0017] In WO2019 / 021600A1 it was disclosed that reduced circulatory levels of bio-ADM were present in- and being predictive for Dementia, especially Alzheimer’s Disease. A model of subcortical vascular dementia was reproduced in mice by placing micro coils bilaterally on the common carotid arteries. Using mice overexpressing circulating ADM, the effect of ADM was assessed on cerebral perfusion, cerebral angioarchitecture, oxidative stress, white matter change, cognitive function, and brain levels of cAMP, vascular endothelial growth factor, and basic fibroblast growth factor. These data indicate that ADM promotes arteriogenesis and angiogenesis, inhibits oxidative stress, preserves white matter integrity, and prevents cognitive decline after chronic cerebral hypoperfusion.

[0018] Thus, ADM may serve as a strategy to tackle subcortical vascular dementia Maki, T. et al.,1122 1128 (2011)).

[0019] The blood-brain barrier (BBB) is a complex dynamic interface that transduces biomechanical and biochemical signals from the vascular system and the brain and is responsible for maintaining homeostasis of the brain by regulating exchange of water, ions, nutrients, metabolites, neurotransmitters, and other cells (e.g., leukocytes), while limiting entry of potentially toxic xenobiotics in the blood. The BBB is formed, in part, by highly specialized endothelial cells that line brain capillaries. The tight junctions formed by brain microvascular endothelial cells (BMECs) regulate paracellular transport, whereas transcellular transport is regulated by specialized transporters, pumps, and receptors. This barrier regulates transport by transducing signals from the vascular system and the central nervous system. Such as the vascular endothelium in general, BBB in particular is disrupted in several pathological conditions, such as, but not limited to, stroke, multiple sclerosis, epilepsy, dementias such as Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), Parkinson’s Disease, Sepsis and severe Sepsis, experimental Colitis, Kidney disorders and others (Demeule, M. et al., Vascul. Pharmacol. 38, 339 348 (2002), Abbott, N. J., Patabendige, et al., Neurobiol. Dis. 37, 13 25 (2010). Daneman, R., Ann. Neurol. 72, 648 672 (2012) and Hernandez, L. et al., Sci. Rep. 12, 4414 (2022)).

[0020] Therefore, it is desired to increase circulating bio-ADM levels in clinical conditions as described above, to facilitate among others the regeneration of the endothelial barrier and / or prevent damage of endothelial barrier.

[0021] Clinical testing of bio-ADM was conducted in cardiovascular indications with a measurable hemodynamic end point such as pulmonary hypertension, hypertension, heart failure and acute myocardial infarction (MCI), IBD and refractory Crohn’s disease (see e.g. Ashizuka, S. et al., Dig. Dis. Sci. 61, 872 880 (2016) and Kataoka, Y. et al., J. Cardiovasc. Pharmacol. 56, 413 419 (2010)). Bio- ADM administration showed improvement of clinical symptoms in several studies in patients suffering from the aforementioned conditions. Pharmacodynamic effects comprised among others lowering of systemic and pulmonary arterial blood pressure and increase of cardiac output, as well as reduction of serum inflammatory cytokines and improved mucosal healing in IBD (see e. g. Troughton, R. W. et al., 588 593 (2000), and Nagaya, N. et al., Peptides 25, 2013 2018 (2004)).

[0022] Thus, the above data indicate that ADM induces favorable hemodynamic, hormonal, and myocardial changes both in animal experimental models and in patients. These effects are likely mediated by the vasodilatory properties of intravascular ADM, although they may be mediated by other mechanisms as well.

[0023] In summary, based on evidence from a wealth of experimental data in animals and clinical trials in humans, elevation of bio-ADM to supraphysiological levels might be considered as a target mechanism for the treatment of a variety of disease conditions in man and animals.

[0024] However, the major limitations of the use of bio-ADM as therapeutic agent is the nature of bio-ADM, which restricts the application of wild-type (wt) bio-ADM, especially in CVDs, to continuous intravenous injection or infusion, mostly accounted to the strong vasoactive properties of bio-ADM, which can cause a systemic drop in vascular tonus. Single-injections of higher doses of bio-ADM lead to a significant drop of mean arterial blood pressure (W02013064508A1), but also an infusion of bio- ADM at a rate of 100 ng / kg / min in an LPS induced endotoxemia model led to a decrease of MABP to 64 mmHg bearing the risk of inducing a circulatory shock (Ertmer, C. et al., Crit. Care 11, 1 (2007) and Burstein, B. et al., Crit. Care 24, 513 (2020)). Another obstacle is the short-lasting nature of achieved effects with an immediate ceasing after the end of administration (Nagata, S. et al., Peptides 121, 170133 (2019)). These findings correlated well with the known pharmacokinetic profile of bio-ADM having a relatively short in vivo half-life of approximately 22 minutes (Dschietzig, T. et al., Biochem. Biophys. Res. Commun. 294, 315 318 (2002)).

[0025] Several strategies were developed with respect to sustainable bio-ADM elevation in the blood-stream, thereby targeting the N-terminus of bio-ADM, since it is unrelated to its biological activity. Administration of Adrecizumab, an N-terminal, non-neutralizing anti-ADM antibody, leads to a sustainable increase of bio-ADM in circulation for up to 15 days, leading to improvement of endothelial function and increasing the chance of a positive outcome in severe sepsis or shock (Deniau, B. et al., Expert Opin. Investig. Drugs 30, 95 102 (2021)). Additionally, a favorable outcome was observed in critically ill COVID-19 and ARDS patients after administration of the N-terminal anti-ADM antibody (Karakas, M. et al., Biomolecules 10, (2020)).

[0026] The surprising finding of this invention is the effective utilization of ADM-Gly as a pharmaceutical agent to elevate the concentration of bio-ADM in the bloodstream. Unlike the administration of bio- ADM, direct administration of ADM-Gly enables a slow and sustained increase in bio-ADM levels in the circulatory system, without inducing a systemic reduction in vascular tone. This phenomenon is rooted in the scientific presumption that the binding affinity of ADM-Gly to the CRLR-receptor is significantly lower compared to that of bio-ADM.

[0027] Another surprising finding of this invention also is the reparative impact on blood-brain barrier integrity observed in the CLP mouse model when ADM-Gly is employed as a therapeutic agent.

[0028] Moreover, the application of ADM-Gly demonstrated no adverse effects on the overall physical condition and behavior of subjects in the CLP model, reinforcing its potential as a safe and effective medicament.

[0029] Subject Matter of the Invention

[0030] Subject matter of the present invention is ADM-Gly for use in treatment of a disease in a subject, wherein ADM-Gly (SEQ ID No.: 4, 7- 12) is in modified or unmodified form.

[0031] For disambiguation, the person skilled in the art recognizes that the subject matter of the present invention is ADM-Gly and / or a fragment thereof for use in treatment of a disease in a subject, wherein ADM-Gly and / or a fragment thereof (SEQ ID No.: 4, 7-12) is in modified or unmodified form.

[0032] ADM-Gly may be the wildtype ADM-Gly (1-53 ADM) (SEQ ID No.: 4), and / or N-terminal truncated fragments thereof (SEQ ID No.: 7-12), and / or modified ADM-Gly, i.e. modified N-terminal truncated fragments thereof (SEQ ID No.: 7-12) or modified wildtype ADM-Gly (1-53 ADM) (SEQ ID No.: 4).

[0033] For disambiguation, the person skilled in the art recognizes that ADM-Gly may be the wildtype ADM- Gly (1-53 ADM) (SEQ ID No.: 4) and / or a N-terminal truncated fragment thereof (SEQ ID No.: 7-12). Therefore, the person skilled in the art recognizes that wildtype ADM-Gly (aa 1-53 of ADM, SEQ ID No. 4) and / or a N-terminal truncated fragment thereof (SEQ ID No.: 7-12) may be modified.

[0034] The person skilled in the art recognizes that in the present invention the wording “ADM-Gly (SEQ ID No.: 4, 7-12)” in particular refers to ADM-Gly (SEQ ID No. 4) and / or a fragment thereof (SEQ ID No.: 7-12). In one embodiment ADM-Gly may be modified with PEG, in particular 1-100 kDa PEG. ADM-Gly maybe modified with albumin, and / or Fc-fragments of IgG’s and / or with XTEN.

[0035] An embodiment of the present invention is ADM-Gly (SEQ ID No. 4) or ADM-Gly fragment (SEQ ID No.: 7-12) ADM-Gly as drug for use in treatment of a disease in a subject, wherein ADM-Gly is in modified or unmodified form (SEQ ID No.: 4, 7-12).

[0036] Modifications of ADM-Gly may be selected from the group comprising:

[0037] • 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.

[0038] • Conjugation and / or bioconjugation of polypeptides of interest with serum proteins, such as albumins or immunoglobulins or parts of immunoglobulins to produce fusion proteins:

[0039] • Fusion of polypeptides with Albumins, e. g. serum Albumin or recombinant serum Albumin.

[0040] • 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.

[0041] • Fusion of polypeptides with IgG Fc regions or Transferrin

[0042] • Post-translational modifications attaching natural or synthetic polymers to the polypeptide of interest. Example for such polymers, but not limited to, are

[0043] ■ PEG, either single-stranded or branched PEG, having varying molecular weights covalently fused to polypeptides of interest.

[0044] ■ 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.

[0045] ■ PAS, which is a peptide polymer consisting of amino acids proline, alanine and serine with 100- 200 PAS repeats forming the polymer.

[0046] ■ 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.

[0047] ■ HAP, which is a repeated sequence of glycine rich (Gly4Ser)n polypeptide, wherein n is inbetween of 100-200 and is covalently attached to the protein of interest.

[0048] ■ 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. ■ 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, Hydroxyethyls (HES), Heparosan (HEP), Hyaluronic acid (HA) represents the attachment of polysaccharides.

[0049] ■ PSA (Polysialic acid), wherein PSA polymers are covalently attached to the polypeptide of interest.

[0050] Modifications of ADM-Gly may also include modifications, which allow ADM-Gly 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. 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 comprises Carrier-linked prodrugs (Carrier prodrugs), Cascade prodrugs and PEG-based carrier prodrugs.

[0051] A carrier-linked prodrug is 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 et al., Nature Reviews Drug discovery, 2008, 7 255-270).

[0052] A PEG-based carrier prodrug may refer to a carrier-linked prodrug wherein the carrier group is PEG. In terms of this invention a PEG-based carrier prodrug of ADM-Gly may mean the addition of a linker structure according to formula 1 to the c-terminus of ADM-Gly (SEQ ID No.: 14, 17-21, 27), wherein R1 represents a C-terminal amino acid of ADM-Gly according to SEQ ID No.: 14, 17-21, 27 as indicated in formula 1 and the linker is further conjugated with linear or branched PEG of 1-100 kDa at positions R2 and / or R3 of the linker according to formula 1 , and wherein n according to formula 1 may be 1 , 2 or 3. For disambiguation, the person skilled in the art recognizes that ADM-Gly (SEQ ID No.: 14, 17-21, 27), refers to ADM-Gly as defined herein, in particular ADM-Gly (SEQ ID No.: 4, 7-12), more particularly ADM-Gly (SEQ ID No. 4) and / or ADM-Gly fragments (SEQ ID No. 7-12) and most particularly ADM-Gly (SEQ ID No. 4).

[0053] The skilled person will readily recognize in view of the purpose of the present invention that PEG is reasonably attached to the N-terminal end of ADM-Gly. Therefore, embodiments of the invention relating to a PEG-based carrier prodrug of ADM-Gly may mean the addition of a linker structure according to formula 1 to the N-terminus of ADM-Gly (SEQ ID No.: 14, 17-21, 27), wherein R1 represents a N-terminal amino acid of ADM-Gly according to SEQ ID No.: 14, 17-21, 27 as indicated in formula 1 and the linker is further conjugated with linear or branched PEG of 1-100 kDa at positions R2 and / or R3 of the linker according to formula 1 , and wherein n according to formula 1 may be 1 , 2 or 3. For disambiguation, the person skilled in the art recognizes that ADM-Gly (SEQ ID No.: 14, 17-21, 27), refers to ADM-Gly as defined herein, in particular ADM-Gly (SEQ ID No.: 4, 7-12), more particularly ADM-Gly (SEQ ID No. 4) and / or ADM-Gly fragments (SEQ ID No. 7-12) and most particularly ADM-Gly (SEQ ID No. 4).

[0054] Formula 1

[0055] The person skilled in the art is aware that also other forms of prodrugs are known in the art. For examples of such prodrugs see: Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985) and Methods in Enzymology, Vol. 42, p. 309-396, edited by K. Widder, et al. (Academic Press, 1985); A Textbook of Drug Design and Development, edited by Krogsgaard-Larsen and H. Bundgaard, Chapter 5 “Design and Application of Prodrugs” by H. Bundgaard p. 113-191 (1991); H. Bundgaard, Advanced Drug Delivery Reviews 8, 1-38 (1992); H. Bundgaard, et al., Journal of Pharmaceutical Sciences, 77, 285 (1988); and N. Kakeya, et al., Chem. Pharm. Bull., 32, 692 (1984).

[0056] Modified ADM-Gly may refer to ADM-Gly (SEQ ID No.: 4, 7-12) 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.

[0057] ADM-Gly modified via PEGylation may refer to ADM-Gly (SEQ ID No.: 4, 7-12) modified with 1+n molecules of polyethylene glycol (PEG), whereas n is an integer in the range of 0 to 10 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 2O.ADM-Gly modified via fusion to native serum Albumin or recombinant human serum Albumin may mean ADM-Gly (SEQ ID No.: 4, 7-12) fused to native human serum Albumin or recombinant human serum Albumin at the N- terminus of ADM-Gly according to (SEQ ID No.: 4, 7-12).

[0058] ADM-Gly modified via fusion to IgG Fc regions may refer toADM-Gly (SEQ ID No.: 4, 7-12) fused to human IgGl Fc Region at the N-terminus of ADM-Gly according to (SEQ ID No.: 4, 7-12).

[0059] ADM-Gly modified via XTEN may refer to ADM-Gly (SEQ ID No.: 4, 7-12) fused to a XTEN moiety at the N-terminus of ADM-Gly according to (SEQ ID No.: 4, 7-12).

[0060] 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 the preferred embodiment fusion may refer to covalent linkage.

[0061] 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.

[0062] In one embodimentADM-Gly (SEQ ID No.: 4, 7-12) modified via fusion to native serum Albumin or recombinant serum Albumin, preferably modified via fusion to IgG Fc regions, more preferably via fusion to XTEN, even more preferably via PEGylation is to be used for treatment of a disease in a subject.

[0063] In another embodiment ADM-Gly (SEQ ID No.: 4, 7-12) modified to function as PEG-based Prodrug for use in treatment of a disease in a subject.

[0064] In the preferred embodiment ADM-Gly (SEQ ID No.: 4, 7-12) modified to function as PEG-based Prodrug for use in treatment of a disease in a subject is modified via PEGylation.

[0065] In terms of this invention, the biological activity of the modified ADM-Gly (SEQ ID No.: 4, 7-12) shall remain present to an extent of at least 1 % (biological activity 5-300%) when directly compared to the non-modified active moiety (SEQ ID No.: 4), preferably of at least 5% (biological activity 5-300%), more preferably of at least 15% (biological activity 15-300%), more preferably of at least 30% (biological activity 30-300%), more preferably of at least 50% (biological activity 50-300%) more preferably of at least 70% (biological activity 70-300%), more preferably of at least 90% (biological activity 90-300%), more preferably of at least 150% (biological activity 1500-300%), more preferably of at least 200% (biological activity 200-300%) and most preferably of at least 250% (biological activity 250 -300%). In terms of ADM-Gly, biological activity shall mean the modified and unmodified ADM- Gly capability to stimulate the ADM natural receptor after the ADM-Gly has been amidated by PAM, which may be tested as described in Example 6 using a distinctive reporter assay. Thereby, binding of amidated Adrenomedullin to the ADM receptor on CHO-K1 cells generates cAMP (cyclic adenosine monophosphate), which is quantified as described in example 6. The amount of generated cAMP by modified ADM-Gly after its amidation, set in relation to wild-type amidated ADM, results in the biological activity.

[0066] Subject matter of the present invention is ADM-Gly for use in treatment of a disease in a subject, wherein ADM-Gly is to be used in said subject in need of elevated level of bio-ADM (SEQ ID No.: 5, 13-18) in the circulation wherein elevated level means by at least 10 pg / mL (10-2000 pg / mL), preferably by at least 20 pg / mL (20-2000 pg / mL), more preferably by at least 30 pg / mL (30-2000 pg / mL), more preferably by at least 40 pg / mL (40-2000 pg / mL), most preferably by at least 50 pg / mL (50-2000 pg / mL).

[0067] For disambiguation, the person skilled in the art recognizes that in the present invention the wording “bio-ADM (SEQ ID No.: 5, 13-18)” preferably refers to bio-ADM (SEQ ID No. 5) and / or a fragment thereof (SEQ ID No.: 13-18). The person skilled in the art also recognizes that if not indicated otherwise, “bio-ADM” preferably also refers to bio-ADM (SEQ ID No. 5) and / or a bio-ADM fragment (SEQ ID No.: 13-18).

[0068] Subject matter of the present invention is ADM-Gly, wildtype (1-53 ADM) (SEQ ID No.: 4), and / or N- terminal truncated fragments thereof (SEQ ID No.: 7-12), and / or modified ADM-Gly, i.e. modified N- terminal truncated fragments thereof (SEQ ID No.: 7-12) or modified wildtype ADM-Gly (1-53 ADM) / SEQ ID No.: 4), for use in treatment of a disease in a subject, wherein said subject is in need of elevated level of bio-ADM in the circulation wherein elevated level means elevation by at least 10 pg / mL (10- 2000 pg / mL), preferably by at least 20 pg / mL (20-2000 pg / mL), more preferably by at least 30 pg / mL (30-2000 pg / mL), more preferably by at least 40 pg / mL (40-2000 pg / mL), most preferably by at least 50 pg / mL (50-2000 pg / mL). An embodiment of the present invention is ADM-Gly, wildtype (1-53 ADM) (SEQ ID No.: 4), and / or N-terminal truncated fragments thereof (SEQ ID No.: 7-12), and / or modified ADM-Gly, i.e. modified N-terminal truncated fragments thereof (SEQ ID No.: 7-12) or modified wildtype ADM- Gly (1-53 ADM) / SEQ ID No.: 4), for use in treatment of a disease in a subject, wherein said subject is in need of elevated level of bio-ADM in the circulation and wherein elevation of bio-ADM may refer to an elevation in the range of 10 - 10000 pg / mL, preferably in the range of 20 6000 pg / mL, more preferably in the range of 30- 4000 pg / mL, more preferably in the range of 40-3000 pg / mL, most preferably in the range of 50-1000 pg / mL.

[0069] In one embodiment the elevation of bio-ADM levels may be determined using an immunoassay, that specifically discriminates between amidated and non-amidated ADM, calibrated with wild-type bio- ADM of known concentration and molecular weight. Said assay might be a radioimmunoassay, a fluorescence-based immunoassay or a competitive assay. In a preferred embodiment, concentration of bio-ADM may be determined using a luminescence-based sandwich immunoassay as described in Example 1. The molar concentration of the bio-ADM calibrator peptide preparation may be calculated from the mass concentration of the peptide preparation using the molecular weight of bio-ADM, wherein the mass concentration results from dissolving a specific mass of the bio-ADM preparation in a defined volume of a solvent. 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.

[0070] Administering the ADM-Gly, wildtype (1-53 ADM) (SEQ ID No.: 4), and / or N-terminal truncated fragments thereof (SEQ ID No.: 7-12), and / or modified ADM-Gly, i.e. modified N-terminal truncated fragments thereof (SEQ ID No.: 7-12) or modified wildtype ADM-Gly (1-53 ADM) / SEQ ID No.: 4), according to the invention leads to a prolonged bioavailability of bio-ADM in the circulation for at least 30 minutes, preferably for at least 1 hour, more preferably for at least 8 hours, more preferably for at least 12 hours, more preferably for at least 24 hours, more preferably for at least 48 hours, most preferably for at least 72 hours after treatment, alternatively wherein said intervention leads to a prolonged bioavailability of bio-ADM in the circulation for 30 minutes, particularly 30 Minutes to 1 hour, more particularly 30 minutes to 8 hours, more particularly 30 minutes to 12 hours, more particularly 30 minutes to 24 hours, more particularly 30 minutes to 48 hours and more particularly 30 minutes to 72 hours after treatment.

[0071] In another embodiment prolonged bioavailability of bio-ADM may refer to a prolongation in the timerange of 30 minutes-336 hours, preferably in the time-range of 1-288 hours, more preferably in the timerange of 8-240 hours, more preferably in the time-range of 12-216 hours, more preferably in the time- range of 24-192 hours, more preferably in the time-range of 48-180 hours and most preferably in the time-range of 72-168 hours.

[0072] In one embodiment, the bioavailability or prolonged bioavailability of bio-ADM levels may be determined using an immunoassay, that specifically discriminates between amidated and non-amidated ADM, calibrated with wild-type bio-ADM of known concentration and molecular weight, where in bio- ADM levels are determined from blood derived samples drawn at specific time-points after administration of said present combination of the invention. Said assay might be a radioimmunoassay, a fluorescence-based immunoassay or a competitive assay.

[0073] In another embodiment, the bioavailability or prolonged bioavailability of ADM-Gly may be determined by using an Immunoassay for determination of the total ADM concentration in the circulation using an antibody combination, that does not react with the c-terminus of ADM.

[0074] Afterwards the known concentration of bio-ADM may be subtracted from the total ADM concentration to result in the concentration of ADM-Gly.

[0075] In a preferred embodiment, concentration of ADM-Gly may be determined using a luminescence-based sandwich immunoassay as described in Example 1, wherein the tracer antibody does not cross-react with bio-ADM and specifically reacts with the glycine-extended C-terminus of ADM-Gly. Using said preferred assay ADM-Gly levels may be determined from blood derived samples drawn at specific timepoints after administration of said present combination of the invention. Specific timepoints may refer to generation of blood-derived samples within of 1 minute-30 minutes, preferably within of 1 minute to 1 hour, more preferably within of 1 minute-8 hours, more preferably within of 1 minute- 12 hours, more preferably within of 1 minute-24 hours, more preferably within of 1 minute-48 hours, more preferably within of 1 minute-72 hours and most preferably within of 1 minute-336 hours after administration of said present combination of the invention. The assay is calibrated with wild-type ADM-Gly of known concentration and molecular weight (Example 1).

[0076] The bioavailability of PAM may be assessed using an activity assay thereby allowing the determination of activity of the enzyme, independent of its molecular weight and therefore independent of any used modification of the enzyme. The activity of an enzyme may be defined in Units or Units / L (Units per Liter of sample), where in units may mean the amount of product formed by an enzyme in a distinct period of time. The activity assay, as described in Example 2, uses wild-type ADM-Gly as a substrate for the PAM-enzyme and wherein the PAM enzyme originates from the used sample material applied in the assay. After conversion of the substrate ADM-Gly to bio-ADM by sample PAM, bio-ADM is quantified, and the activity of the PAM enzyme is calculated as Units (amount of bio-ADM formed per time). 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. 29 - 34; SEQ ID Nos. 19 - 24 show the respective prepro-forms).

[0077] 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. JNeurosci 12(1): 222 34).

[0078] The precursor protein of the largest known PAM Isoform 1 (SEQ ID No. 19) consists of 973 amino acids. 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. JNeurosci Res 88(12);2535-45). The sequences of PAM isoforms 1 to 6 after cleavage of the N-terminal signal sequence or pre-peptide (amino acid 1-20) as well as the pro-region or pro-peptide (amino acids 21 to 30) are given for disambiguation as SEQ ID No. 29 to 34.

[0079] The PAM 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 pro-region or pro-peptide (within amino acids 31-494 of isoform 1 (SEQ ID No. 25)). The second catalytic activity (PAL) is located after exon 16 in isoform 1 within amino acids 495-817 (SEQ ID No. 26).

[0080] 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. 29, 30, 33 and 34) as well within of one polypeptide as a soluble protein lacking the transmembrane domain (TMD) (isoforms 3 and 4; corresponding to SEQ ID No. 31 and 32). 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). In terms of the present invention, bioavailability may refer to the increase of the concentration of bio- ADM in circulation after administration of said compound.

[0081] The term prolonged bioavailability may refer to an increase of the concentration of bio-ADM or total ADM over a distinct period of time. In terms of the present invention, prolonged bioavailability of ADM-Gly or bio-ADM, may refer to the elevation of concentration of bio-ADM or ADM-Gly in circulation for at least 30 minutes, preferably for at least 1 hour, more preferably for at least 8 hours, more preferably for at least 12 hours, more preferably for at least 24 hours, more preferably for at least 48 hours, most preferably for at least 72 hours after treatment. In an embodiment of the present invention prolonged bioavailability of bio-ADM and / or ADM-Gly may refer to a prolongation in the time-range of 30 minutes-336 hours, preferably in the time-range of 1-288 hours, more preferably in the time-range of 8-240 hours, more preferably in the time-range of 12-216 hours, more preferably in the time-range of 24-192 hours, more preferably in the time-range of 48-180 hours and most preferably in the time-range of 72-168 hours.

[0082] Subject matter of the present invention is ADM-Gly for use in treatment of a disease in a subject, wherein said disease is characterized by a ADM-Gly / bio-ADM ratio above a threshold in a sample of bodily fluid of said subject and / or a bio-ADM level below a certain threshold and / or a ADM-Gly level below a certain threshold and / or a ADM-Gly level above a certain threshold in combination with level of PAM and / or its isoforms and / or fragments thereof below a threshold and / or

[0083] -a level of PAM and / or its isoforms and / or fragments thereof below a threshold.

[0084] Subject matter of the present invention is ADM-Gly, wildtype ADM-Gly (1-53 ADM) (SEQ ID No.: 4), and / or N-terminal truncated fragments thereof (SEQ ID No.: 7-12), and / or modified ADM-Gly, i.e. modified N-terminal truncated fragments thereof (SEQ ID No.: 7-12) or modified wildtype ADM-Gly (1-53 ADM) / SEQ ID No.: 4), for use in treatment of a disease in a subject, wherein said disease is characterized by an ADM-Gly / bio-ADM ratio above a threshold in a sample of bodily fluid of said subject and / or a bio-ADM level below a certain threshold and / or an ADM-Gly level below a certain threshold and / or an ADM-Gly level above a certain threshold together with the level of PAM and / or its isoforms and / or fragments thereof below a threshold and / or a level of PAM and / or its isoforms and / or fragments thereof below a threshold.

[0085] Subject matter according to the invention is ADM-Gly (SEQ ID No.: 4, 7-12) for use in treatment of a disease in a subject, wherein the molar ratio of ADM-Gly and bio-ADM (SEQ ID No.: 5, 13-18) (ADM- Gly / bio-ADM) in a bodily fluid is elevated over 1.5, preferably elevated over 1.6, more preferably elevated over 1.7, more preferably is elevated over 1.8, more preferable is elevated over 1.9, more preferably is higher than 2 and / or the concentration of PAM (SEQ ID No. 19-28) in a bodily fluid is equal or below 100 ng / mL (100 ng / mL to 0 ng / mL), preferably equal or below 90 ng / mL (90 ng / mL to 0 ng / mL), more preferably equal or below 80 ng / mL (80 ng / mL to 0 ng / mL), more preferably equal or below 60 ng / mL (60 ng / mL to 0 ng / mL), more preferably equal or below 50 ng / mL (50 ng / mL to 0 ng / mL) and most preferably equal or below 40 ng / mL (40 ng / mL to 0 ng / mL) and / or the activity of PAM (SEQ ID No. 19-28) in a bodily fluid is equal or below 24 Units / L, more preferably equal or below 20 Units / L, more preferably equal or below 16 Units / L, more preferably equal or below 14 Units / L, and most preferably equal or below 10 Units / L and / or bio-ADM (SEQ ID No.: 5, 13-18) is equal or below 20 pg / mL (20 pg / mL-0 pg / mL), preferably equal or below 15 pg / mL (15 pg / mL 0 pg / mL), preferably equal or below 10 pg / mL (10 pg / mL-0 pg / mL), preferably equal or below 5 pg / mL (5 pg / mL - 0 pg / mL) and / or

[0086] ADM-Gly (SEQ ID No.: 4, 7-12) is equal or above 15 pg / mL, preferably equal or above 20 pg / mL, preferably equal or above 30 pg / mL, preferably is equal or above 50 pg / mL, together with the concentration of PAM (SEQ ID No. 19-28) in a bodily fluid is equal or below 100 ng / mL (100 ng / mL to 0 ng / mL), preferably equal or below 90 ng / mL (90 ng / mL to 0 ng / mL), more preferably equal or below 80 ng / mL (80 ng / mL to 0 ng / mL), more preferably equal or below 60 ng / mL (60 ng / mL to 0 ng / mL), more preferably equal or below 50 ng / mL (50 ng / mL to 0 ng / mL) and most preferably equal or below 40 ng / mL. (40 ng / mL to 0 ng / mL).

[0087] For disambiguation, the person skilled in the art recognizes that “PAM (SEQ ID No.: 19 to 28)” or “PAM” preferably refers to PAM and / or an isoform of PAM (as comprised in the prepro sequences SEQ ID No. 19-24; for sake of disambiguation the mature PAM sequences are shown in SEQ ID No. 29-34) and / or a fragment of PAM (SEQ ID No. 25-28). An embodiment of the present invention is ADM-Gly (SEQ ID No.: 4, 7-12) for use in treatment of a disease in a subject, wherein the given levels of ADM-Gly may refer to a range of 15 10000 pg / mL, preferably to 20 8000 pg / mL, more preferably to 30- 5000 pg / mL, and most preferably in the range of 50-4500 pg / mL.

[0088] Another embodiment of the present invention is ADM-Gly (SEQ ID No.: 4, 7-12) for use in treatment of a disease in a subject, wherein ADM-Gly (SEQ ID No.: 4, 7-12) is below 15 pg / mL (15 pg / mL - 0 pg / mL), preferably below 10 pg / mL (10 pg / mL - 0 pg / mL), preferably below 5 pg / mL (5 pg / mL - 0 pg / mL).

[0089] Subject matter of the present invention is ADM-Gly for use in treatment of a disease in a subject according to any of claims 1 to 3, wherein ADM-Gly is to be used in treatment of the diseases, selected from the group comprising pulmonary hypertension and hypertension, type-2 diabetes, inflammatory bowel disease and / or Crohn’s disease, sepsis and / or septic shock, cerebral infarction, mild cognitive impairment (MCI) and Alzheimer’s Disease (AD).

[0090] Subject matter according to the invention is ADM-Gly, wildtype ADM-Gly (1-53 ADM) (SEQ ID No.: 4), and / or N-terminal truncated fragments thereof (SEQ ID No.: 7-12), and / or modified ADM-Gly, i.e. modified N-terminal truncated fragments thereof (SEQ ID No.: 7-12) or modified wildtype ADM-Gly (1-53 ADM) / SEQ ID No.: 4) for use in treatment of a disease in a subject, wherein said disease is selected from the group comprising cardiovascular diseases, kidney / water electrolyte system diseases, brain and neurological diseases, especially dementia, genitourinary disease, gastrointestinal diseases, orthopedic disease, endocrine and metabolic disorders, respiratory disorders, immune disorders and / or inflammatory or infectious diseases.

[0091] An embodiment of the present invention is ADM-Gly, wildtype ADM-Gly (1-53 ADM) (SEQ ID No.: 4), and / or N-terminal truncated fragments thereof (SEQ ID No.: 7-12), and / or modified ADM-Gly, i.e. modified N-terminal truncated fragments thereof (SEQ ID No.: 7-12) or modified wildtype ADM-Gly (1-53 ADM) / SEQ ID No.: 4) for use in treatment of a disease in a subject wherein said disease is selected from the group comprising pulmonary hypertension and hypertension, type-2 diabetes, inflammatory bowel disease and / or Crohn’s disease, sepsis and / or septic shock, cerebral infarction, mild cognitive impairment (MCI) and Alzheimer’s Disease (AD).

[0092] The blood brain barrier (BBB) is a continuous endothelial membrane within brain microvessels that has sealed cell-to-cell contacts, and is sheathed by mural vascular cells and perivascular astrocyte end-feet. There is an extensive body of evidence from the literature which shows that bio-ADM is indispensable for an intact endothelial and blood-brain barrier function and that administration of bio-ADM and other peptide hormones to supra-physiological levels has protective effects towards endothelial and bloodbrain barrier function (for review Kis B et al.. Peptides. 2006 Jan;27(l):211-22) The BBB protects neurons from factors present in the systemic circulation, and maintains the highly regulated CNS internal milieu, which is required for proper synaptic and neuronal functioning. BBB disruption allows influx into the brain of neurotoxic blood-derived debris, cells, and microbial pathogens, and is associated with inflammatory and immune responses, which can initiate multiple pathways of neurodegeneration (Sweeney et al., Nat. Rev. Neurol. 14, 133 150 (2018)). BBB dysfunction occurs in several diseases, including MS, epilepsy, stroke and dementias, including Alzheimer’s disease (AD). In these conditions, BBB dysfunction plays a central role in the pathology, as BBB disruption leads to ion dysregulation, edema, neuroinflammation, culminating in neuronal dysfunction, increased intracranial pressure and neuronal degeneration (profaci nature).

[0093] The pharmaceutical composition according to the present invention is capable of treating and / or preventing BBB dysfunction and dysregulation as outlined in Example 5 and therefore may be used for treatment and / or prevention of diseases associated with a BBB dysfunction and / or disruption, which specifically include, but are not limited to, brain and neurological diseases such as cerebral infarction, mild cognitive impairment, dementia, cerebrovascular dementia, Alzheimer's disease, and encephalitis.

[0094] The impairment and / or disruption and / or increased permeability of BBB may be quantified in a patient using advanced clinical imaging technologies including, but not limited to, Dynamic Contrast-Enhanced MRI (DCE-MRI), Dynamic Susceptibility Contrast Perfusion Imaging (DSC-MRI), Glucose Chemical Exchange Saturation Transfer Imaging (GlucoCEST), Arterial Spin Labelling (ASL), Intravoxel Incoherent Motion Imaging (IVIM). The advanced clinical imaging techniques may help identify patients with impaired BBB in clinical settings including, but not limited to, acute stroke, Cerebral small vessels disease and dementias, such as mild cognitive impairment (MCI) and Alzheimer’s Disease (AD) (reviewed in: Chassidim, Y. et al., Fluids Barriers CNS 10, 9 (2013) and Elschot EP et al., Invest Radiol. 2021 Jan;56(l)).

[0095] The major component of BBB is the endothelium involved not only in the formation of BBB in particular, but also in the formation of the vascular barrier in general. Blood-brain barrier and vascular barrier dysfunction or endothelial dysfunction is a systemic pathological state of the endothelium and can be broadly defined as an imbalance between vasodilating and vasoconstricting substances produced by and / or acting on the endothelium (Deanfield J et al., J Hypertens. 2005 Jan;23(l):7-17). Normal functions of endothelial cells include mediation of coagulation, platelet adhesion, immune function and control of volume and electrolyte content of the intravascular and extravascular spaces. The endothelium is a cellular monolayer that lines the entire cardiovascular system and regulates many processes including vascular tone, thrombosis, angiogenesis, and inflammation. Endothelial cells have been shown to be phenotypically dynamic and, in response to a variety of local and systemic stimuli, are able to transition between quiescent and activated states (Colombo PC et al., Curr Heart Fail Rep. 2015 Jun;12(3):215-22).). In recent years, emerging research has demonstrated that endothelial dysfunction is a major contributor to cardiovascular disease, including hypertension, atherosclerosis, and congestive heart failure (Gutierrez E, et al., Eur Heart J. 2013 Nov; 34(41):3175-81).

[0096] The endothelium tightly controls the exchange of fluid from the circulation to the surrounding tissues and dysfunction of this barrier leads to uncontrolled fluid extravasation that may result in congestion and / or edema. A common feature of edema (e. g. pulmonary edema) is increased permeability to water low molecular weight solutes (Rocker GM, et al., Pulmonary vascular permeability to transferrin in the pulmonary oedema of renal failure. Thorax. 1987 Aug;42(8): 620-3).). Endothelial dysfunction can result from and / or contribute to several disease processes, as occurs in hypertension, hypercholesterolaemia, diabetes or septic shock. Endothelial dysfunction is a major pathophysiological mechanism that leads towards coronary artery disease, and other atherosclerotic diseases. Therefore, the application of ADM-Gly as drug, which leads to an increase of bio-ADM, in this invention may be used for treatment and / or prevention of diseases associated with an endothelial and / or vascular dysfunction and / or disruption.

[0097] The pharmaceutical compound of this invention containing ADM-Gly, wildtype ADM-Gly (1-53 ADM) (SEQ ID No.:4) and / or modifications of thereof, e.g. N-terminal truncated fragments thereof (SEQ ID No.: 7-12), and / or modified ADM-Gly, i.e. modified N-terminal truncated fragments thereof (SEQ ID No.: 7-12) or modified wildtype ADM-Gly (1-53 ADM; SEQ ID No.: 4) of one aspect of the present invention, a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient has various symptoms prevented or treated by bio-ADM. Diseases and / or disorders can be prevented or treated as well. The symptoms, diseases and / or disorders include, but are not limited to, the following:

[0098] (1) Brain and neurological diseases: cerebral infarction, mild cognitive impairment, dementia, cerebrovascular dementia, Alzheimer's disease, and encephalitis.

[0099] (2) Infectious diseases caused by infectious organisms such as bacteria, viruses, fungi or parasites, more particularly by infectious bacteria, particularly diseases selected from the group comprising SIRS, sepsis, and septic shock.

[0100] (3) Gastrointestinal diseases: inflammatory diseases (e.g., inflammatory bowel disease or Crohn's disease), ulcerative diseases (e.g., ulcerative colitis), intestinal Behcet's disease, hepatitis, liver fibrosis, cirrhosis, and liver failure.

[0101] (4) Endocrine and metabolic disorders: diabetes and diabetic organ disorders (e.g., diabetic nephropathy or diabetic retinopathy).

[0102] (5) Cardiovascular disease: heart failure, pulmonary hypertension, arteriosclerosis obliterans, Buerger's disease, myocardial infarction, lymphedema, Kawasaki disease, myocarditis, arrhythmia (for example, arrhythmia after catheter ablation surgery), atrial fibrillation, Aortitis, pulmonary hypertension, hypertension, organ damage due to hypertension, peripheral vascular disease, and arteriosclerosis.

[0103] For disambiguation, the person skilled in the art recognizes that the “pharmaceutical compound” is comprised within the definition of the “pharmaceutical composition” of the invention. Accordingly, in embodiments of the present invention, the pharmaceutical composition comprises ADM-Gly. In other embodiments of the present invention, the pharmaceutical composition comprises ADM-Gly and / or an ADM-Gly fragment (SEQ ID No.: 4, 7-12). In further embodiments the pharmaceutical composition preferably comprises the wildtype ADM-Gly (1-53 ADM) (SEQ ID No.: 4) and / or a N-terminal truncated fragment thereof (SEQ ID No.: 7-12). In further embodiments, the pharmaceutical composition comprises modified ADM-Gly (aa 1-53 of ADM, SEQ ID No. 4) and / or a modified N- terminal truncated fragment thereof (SEQ ID No.: 7-12).

[0104] (1) Brain / neurological disorders prevented or treated by the pharmaceutical composition according to an embodiment of the present invention is dementia, in particular mild cognitive impairment, cerebral infarction dementia or Alzheimer’s disease.

[0105] Dementia is a 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. The different causes (sometimes referred to as subtyping) of dementia syndrome are: Alzheimer’s disease (about 50% of cases), vascular dementia (about 25%), mixed Alzheimer’s disease and vascular dementia (included in the above, 25%), Lewy body dementia (15%) and others (about 5% combined) including frontotemporal dementia, focal dementias (such as progressive aphasia), subcortical dementias (such as Parkinson’s disease dementia), and secondary causes of dementia syndrome (such as intracranial lesions).

[0106] Alzheimer's disease (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.

[0107] Mild cognitive impairment (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% 15% 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.

[0108] In a preferred embodiment 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. 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 et al., Increased blood-brain barrier permeability is associated with dementia and diabetes but not amyloid pathology or APOE genotype. Neurobiol Aging. 2017 Mar;51: 104-112.). Additional symptoms may include fluctuations in alertness, visual hallucinations, slowness of movement, trouble walking, and rigidity. 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.

[0109] 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.

[0110] 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., Neurobiol Aging. 2017 Mar;51 : 104-112).m& 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. Neurodegener Dis Manag 4(6): 439 454. Differentiating the different dementia syndromes can be challenging, due to the frequently overlapping clinical features and related underlying pathology. 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).

[0111] 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.

[0112] 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 as outlined above have a common denominator of BBB disruption and / or dysregulation. Therefore “dementia” may refer to Frontotemporal dementia, vascular dementia, Lewy body dementia, Alzheimer’s Dementia and Mild Cognitive Impairment and the pharmaceutical composition according to the present invention may be used for treatment and / or prevention of dementia by protecting the BBB from dysregulation as shown e.g. Example 5.

[0113] (2) Infectious diseases prevented or treated by the pharmaceutical composition according to an embodiment of the present invention are, in particular, sepsis or septic shock.

[0114] The endothelium is an active contributor to sepsis and as such represents a major target for therapy. During sepsis, endothelial cells amplify the immune response and activate the coagulation system. They are both a target and source of inflammation and serve as a link between local and systemic immune responses. In response to cytokines produced by immune cells, the endothelium expresses adhesion molecules and produces vasoactive compounds, inflammatory cytokines, and chemoattractants (Dolmatova EV et al., Cardiovasc Res. 2021 Jan l;117(l):60-73. Hemodynamic instability plays an important role in the development of sepsis and / or septic shock and arises due to a combination of sepsis- induced vasodilation and vascular leakage, the latter of which is caused by disrupted endothelial integrity. Extensive changes occur in the endothelium as a result of circulating damage-associated molecular patterns and pathogen-associated molecular patterns that activate inflammatory and coagulation pathways during sepsis. In turn, this can result in increased leukocyte adhesion, a procoagulant state, vasodilation and endothelial cell permeability, and ultimately widespread edema, shock and lethal organ dysfunction (Geven C. et al., Effects of the Humanized Anti-Adrenomedullin Antibody Adrecizumab (HAM8101) on Vascular Barrier Function and Survival in Rodent Models of Systemic Inflammation and Sepsis. Shock. 2018 Dec;50(6):648-654.). As shown in example 5, the pharmaceutical composition according to the present invention may be used for protecting the endothelial barrier from dysregulation and / or prevent endothelial permeability. (3) The gastrointestinal diseases prevented or treated by the pharmaceutical composition according to an embodiment of the present invention are, in particular, inflammatory diseases (e.g., inflammatory bowel disease (IBD) or Crohn's disease), ulcerative diseases (e.g., ulcerative colitis) or intestinal Bechet's disease.

[0115] Inflammatory bowel diseases (IBD) include Crohn’s disease (CD), ulcerative colitis (UC) (and indeterminate colitis), which share several inflammatory characteristics with other chronic immune disturbances including immune activation, leukocyte infiltration into tissues and increased vascular density. The maintenance of normal vascular barrier supports nutrient and 02 exchange, osmotic balance and leukocyte abundance in the extracellular compartment. In IBD, increased vascular permeability leads to tissue edema and damage in both human IBD and animal models of IBD. This alteration in solute permeability of the vasculature is not restricted to the gut microcirculation but is widespread affecting the vasculature of other organs including the brain. {Cromer WE et al., Role of the endothelium in inflammatory bowel diseases. World J Gastroenterol. 2011 Feb 7;17(5):578-93). The pharmaceutical composition according to the present invention may be used for protecting the vascular barrier from dysregulation and / or prevent vascular permeability as shown in example 5.

[0116] 4) The endocrine and metabolic disorders prevented or treated by the pharmaceuticals of this embodiment are, in particular, diabetes and diabetic organ disorders (e.g., diabetic nephropathy or diabetic retinopathy).

[0117] (5) Cardiovascular diseases prevented or treated by the pharmaceutical composition according to an embodiment of the present invention are, in particular, heart failure, myocardial infarction (e.g., acute myocardial infarction), arrhythmia (e.g., arrhythmia after catheter ablation surgery), atrial fibrillation, pulmonary hypertension or peripheral. It is a vascular disease.

[0118] The state of the art discloses the use of modified bio-ADM, for preventing or treating inflammatory bowel disease by administering said peptides into circulation and thereby elevating the concentration of said peptides in circulation. Thereby either modified or non-modified forms of ADM-Gly (SEQ ID No.: 4, 7-12) are claimed. Thereby the modifications comprise i.a. replacement of the disulfide bond in ADM, but no N-terminal attachments.

[0119] Subject matter of the present invention is ADM-Gly for use in treatment of a disease in a subject, wherein ADM-Gly is to be used in combination with Vitamin C.

[0120] An embodiment of the present invention is ADM-Gly for use in treatment of a disease in a subject, wherein ADM-Gly is to be used in combination with Vitamin C or alone. Thus, subject matter of the present invention is also a ADM-Gly for use in treatment of IBD, whereinADM-Gly (SEQ ID No.: 4, 7-12) is to be used in combination with Vitamin C or alone.

[0121] The state of the art discloses the use of PEG based prodrugs of bio-ADM, to treat or prevent diseases, such as cardiovascular, edematous and / or inflammatory diseases. The prodrug thereby should ensure a slow release of the biologically active compound.

[0122] The state of the art discloses biologically active, stabilized compound of bio-ADM, to treat or prevent diseases, such as cardiovascular, edematous and / or inflammatory diseases. The stabilization of bio- ADM is achieved by modification of bio-ADM with, but not limited to, a PEG moiety, a C3-C100 carboxylic acid moiety, an albumin moiety and others, optionally including a linker region.

[0123] In the state of the art, it was shown that the known effects of bio-ADM in relevant disease models may be reached using at least some of the claimed modified forms of bio-ADM.

[0124] Those effects are reduction of pulmonary arterial pressure and decrease of pulmonary edema, improvement of arterial oxygenation and reduction of vascular leakage and / or hyperpermeability in vivo and in vitro. Such endpoints are relevant for assessing drug candidates effect with respect to treatment and / or prevention of cardiovascular, edematous and / or inflammatory diseases, such as sepsis or septic shock.

[0125] The subject matter of the present invention is in one embodiment a ADM-Gly for use in treatment and / or prevention of cardiovascular, edematous and / or inflammatory diseases, such as sepsis or septic shock, wherein ADM-Gly (SEQ ID No.: 4, 7-12) is to be used in combination with Vitamin C or alone, wherein administered ADM-Gly and / or fragments thereof are amidated by native endogenous PAM enzyme in vivo.

[0126] The state of the art discloses the use of modified bio-ADM as a long-acting ADM derivative for prevention or treatment of i.a. cardiovascular, inflammatory or peripheral vascular diseases. Thereby ADM is N-terminally modified with a palmitoyl group or a polyethylene glycol (PEG) group, while the modification may be linked to the peptide via a linker group. In the state of the art, it was shown that the known effects of bio-ADM in relevant disease models may be reached using at least some of the claimed modified forms of bio-ADM. Those effects are reduction of pulmonary arterial pressure and decrease of pulmonary edema, improvement of arterial oxygenation and reduction of vascular leakage and / or hyperpermeability in vivo. Such endpoints are relevant for assessing drug candidates effect with respect to treatment and / or prevention of cardiovascular, edematous and / or inflammatory diseases, in particular sepsis or septic shock. The state of the art discloses the use of modified bio-ADM as a prophylactic or therapeutic agent for treating or preventing a neurodegenerative disease with abnormal protein accumulation. Thereby either modified or non-modified forms of ADM, or peptides with ADM activity or salts thereof are claimed. The modifications comprise, i.a. a palmitoyl group or a polyethylene glycol (PEG) group in the range of 1-100 kDa. In the state of the art the effect of claimed modified forms of bio-ADM in an Alzheimer’s model in mice was analyzed with respect to short-term memory. The analyzed modified forms of bio-ADM showed improvement of short-term memory in said model.

[0127] The subject matter of the present invention is also a ADM-Gly for use in treatment and / or or preventing a neurodegenerative disease with abnormal protein accumulation, wherein ADM-Gly (SEQ ID No.: 4, 7-12) is to be used in combination with Vitamin C or alone, wherein administered ADM-Gly is amidated by native body own PAM in vivo.

[0128] The surprising and unexpected effect of said combination is the prolonged elevation of resulting bio- ADM in circulation in comparison to the short bioavailability of wi / d / ype-bio-ADM when administered alone. Therefore, said compositions or combinations as the subject matter of the present invention are suitable for treating or preventing a neurodegenerative disease with abnormal protein accumulation.

[0129] The state of the art discloses novel long-lasting bio-ADM, derivatives and fragments and / or modifications thereof, whereas the peptide is modified with serum albumin, that may be linked to the peptide with a linker. Further the use of the described ADM derivatives for preventing or treating of several diseases, such as cardiovascular diseases, Kidney, brain and neurological diseases, genitourinary and gastrointestinal diseases, orthopedic diseases, endocrine and metabolic disorders, respiratory disorders, immune disorders and also other diseases, such as sepsis, shock autoimmune or wound healing diseases is claimed. In the state of the art, it was shown that the known effects of bio-ADM in relevant disease models may be reached using at least some of the claimed modified forms of bio-ADM. Those effects are reduction of systolic and diastolic blood-pressure and reduction of severeness of induced colitis, both in models in rats. Such endpoints are relevant for assessing a drug candidates effect with respect to treatment and / or prevention of cardiovascular, and / or inflammatory diseases of the gastrointestinal tract.

[0130] The subject matter of the present invention is also a ADM-Gly for use in treatment and / or or preventing a cardiovascular, and / or inflammatory diseases of the gastrointestinal tract, wherein ADM-Gly (SEQ ID No.: 4, 7-12) is to be used in combination with Vitamin C or alone, wherein administered ADM-Gly is amidated by native endogenous PAM in vivo leading to a controlled elevation of bio-ADM in the patient’s bloodstream regulated by the amount of endogenous PAM enzyme released into the bloodstream. The surprising and unexpected effect of said combination is the protective and reparative effect on the blood-brain barrier endothelium, as shown in example 5. Therefore, said combinations as the subject matter of the present invention are suitable for treating or preventing a cardiovascular, and / or neurological and / or inflammatory diseases.

[0131] The pharmaceutical composition according to the present invention is a medication that surprisingly allows for the use of non-active precursor of bio-ADM, modified and non-modified wild-type ADM- Gly and fragments thereof, which allows a continuous enrichment of bio-ADM and fragments with bio- ADM activity in circulation. The pharmaceutical composition of the present invention is a medication that allows for treating or preventing of diseases such as brain / neurological disorders, infectious and inflammatory diseases selected from the group comprising SIRS, sepsis, and septic shock, Gastrointestinal diseases including inflammatory diseases such as inflammatory bowel disease or Crohn's disease and ulcerative diseases, endocrine and metabolic disorders, such as diabetes and diabetic organ disorders, cardiovascular diseases such as heart failure, pulmonary hypertension, arteriosclerosis obliterans, myocardial infarction, lymphedema, Kawasaki disease, myocarditis, arrhythmia, atrial fibrillation, aortitis, pulmonary hypertension, hypertension, organ damage due to hypertension, peripheral vascular disease, and arteriosclerosis, by a continuous and controllable release of bioactive peptide hormones as shown in Example 4, bio-ADM, from their inactive precursors, ADM-Gly. As shown in Example 5, the pharmaceutical composition of the present invention, in particular comprising ADM-Gly, is surprisingly capable of treating and / or preventing endothelial dysfunction shown via the example of BBB-disruption prevention and is therefore suitable for treating and / or preventing a large number of diseases associated with endothelial- and / or blood-brain barrier dysfunctions, as, but not limited to, specified above. Another surprising discovery is that the use of inactive ADM-Gly precursor exhibits no adverse effects, such as e.g. a systemic reduction in vascular tone when bio-ADM is administered as a drug.

[0132] The pharmaceutical composition according to the present invention is a medication that is preferably used for the prevention or treatment of symptoms, diseases, and / or disorders as described above (e.g., cardiovascular disease, brain and nerve disorders, or endocrine and gastrointestinal disorders).

[0133] In one embodiment of the present invention, the pharmaceutical composition is used to treat and / or prevent cardiovascular diseases such as heart failure, pulmonary hypertension, arteriosclerosis obliterans, Buerger's disease, myocardial infarction, lymphedema, Kawasaki disease, myocarditis, arrhythmia (for example, arrhythmia after catheter ablation surgery), atrial fibrillation, Aortitis, hypertension, organ damage due to hypertension, peripheral vascular disease, and arteriosclerosis.

[0134] In the preferred embodiment of the present invention, the pharmaceutical composition is used to treat and / or prevent heart failure, pulmonary hypertension, hypertension, organ damage due to hypertension, peripheral vascular disease, and arteriosclerosis.

[0135] In the most preferred embodiment of the present invention, the pharmaceutical composition is used to treat and / or pulmonary hypertension and hypertension.

[0136] In one embodiment of the present invention, the pharmaceutical composition is used to treat and / or prevent endocrine and metabolic disorders such as diabetes and diabetic organ disorders (e.g., diabetic nephropathy or diabetic retinopathy).

[0137] In the preferred embodiment of the present invention, the pharmaceutical composition is used to treat and / or prevent type-2 diabetes. In one embodiment of the present invention, the pharmaceutical composition is used to treat and / or prevent gastrointestinal diseases such as inflammatory diseases (e.g. inflammatory bowel disease or Crohn's disease), ulcerative diseases (e.g. ulcerative colitis), intestinal Behcet's disease, hepatitis, liver fibrosis, cirrhosis, and liver failure.

[0138] In the preferred embodiment of the present invention, the pharmaceutical composition is used to treat and / or prevent inflammatory diseases such as inflammatory bowel disease or Crohn's disease, ulcerative diseases such as ulcerative colitis and / or intestinal Behcet's disease.

[0139] In the most preferred embodiment of the present invention, the pharmaceutical composition is used to treat and / or prevent inflammatory bowel disease and / or Crohn's disease.

[0140] In one embodiment of the present invention, the pharmaceutical composition is used to treat and / or prevent infectious diseases caused by infectious organisms such as bacteria, viruses, fungi or parasites, more particularly by infectious bacteria, particularly diseases selected from the group comprising SIRS, sepsis, and septic shock.

[0141] In the preferred embodiment of the present invention, the pharmaceutical composition is used to treat and / or prevent sepsis and / or septic shock.

[0142] In one embodiment of the present invention, the pharmaceutical composition is used to treat and / or prevent brain and neurological diseases such as cerebral infarction, mild cognitive impairment, dementia, cerebrovascular dementia, Alzheimer's disease, and encephalitis.

[0143] In the preferred embodiment of the present invention, the pharmaceutical composition is used to treat and / or prevent cerebral infarction.

[0144] In the more preferred embodiment of the present invention, the pharmaceutical composition is used to treat and / or prevent dementias, including mild cognitive impairment, cerebrovascular dementia, and Alzheimer’s disease.

[0145] In the most preferred embodiment of the present invention, the pharmaceutical composition of the present invention is used for the prevention and / or treatment of symptoms associated with mild cognitive impairment (MCI) and Alzheimer’s Disease (AD).

[0146] In one embodiment of the present invention, the pharmaceutical composition is used to treat and / or prevent pathological disorders associated with endothelial and / or blood-brain barrier disorders.

[0147] In the preferred embodiment of the present invention, the pharmaceutical composition is used to treat and / or endothelial and / or blood-brain barrier disfunction associated with sepsis or septic shock.

[0148] In the more preferred embodiment of the present invention, the pharmaceutical composition is used to treat and / or prevent endothelial and / or blood-brain barrier dysfunction associated with cerebral infarction. In the more preferred embodiment of the present invention, the pharmaceutical composition is used to treat and / or prevent endothelial and / or blood-brain barrier dysfunction associated with dementias, including mild cognitive impairment, cerebrovascular dementia, and Alzheimer’s disease.

[0149] In the most preferred embodiment of the present invention, the pharmaceutical composition is used to treat and / or prevent endothelial and / or blood-brain barrier dysfunction associated with mild cognitive impairment (MCI) and Alzheimer’s Disease (AD).

[0150] More preferably, it is used for the prevention or treatment of heart failure, acute myocardial infarction, arrhythmia, atrial fibrillation, pulmonary hypertension, peripheral vascular disease, stroke, dementia, inflammatory bowel disease, Crohn's disease, ulcerative colitis, intestinal Behcet's disease, diabetes, diabetic nephropathy, diabetic retinopathy, pulmonary fibrosis, sepsis, or septic shock. By using the drug of this embodiment for the prevention or treatment of the symptoms, diseases and / or disorders described above, it is possible to exhibit superior pharmacokinetics compared to naturally occurring bio- ADM, and the preventive or therapeutic effect is substantially equivalent or superior to that of naturally occurring bio-ADM.As used herein, "prevention" means substantially preventing the occurrence (onset or manifestation) of symptoms, diseases and / or disorders. Also, as used herein, "treatment" means suppressing (e.g., suppressing progression), alleviating, repairing and / or curing symptoms, diseases and / or disorders that have occurred (onset or manifestation).

[0151] Subject matter according to the invention is ADM-Gly for use in treatment of a disease in a subject, wherein ADM-Gly (SEQ ID No.: 4, 7-12) is to be used with Vitamin C.

[0152] An embodiment of the present invention is ADM-Gly for use in treatment of a disease in a subject, wherein ADM-Gly is to be used in combination with Vitamin C and wherein ADM-Gly and Vitamin C are to be administered consecutively, or ADM-Gly and Vitamin C are to be administered simultaneously.

[0153] The threshold according to the present invention may be predetermined by measuring, such as the level of ADM and / or its isoforms and / or fragments in a samples obtained from a cohort of subjects and calculating e.g. the 25th percentile, more particularly the 10th percentile, even more particularly the 5th percentile to define the threshold by which subjects are characterized as suffering from a medical condition or as being at risk of getting a medical condition. In the following examples for thresholds according to the present invention are provided, however, the person skilled in the art is aware on how to determine a specific threshold.

[0154] Subject matter according to the invention is ADM-Gly (SEQ ID No.: 4, 7-12) for use in treatment of a disease in a subject, wherein the molar ratio of ADM-Gly and bio-ADM (SEQ ID No.: 5, 13-18) (ADM- Gly / bio-ADM) in a bodily fluid is elevated over 1.5, preferably elevated over 1.6, more preferably elevated over 1.7, more preferably is elevated over 1.8, more preferable is elevated over 1.9, more preferably is higher than 2 and / or the concentration of PAM (SEQ ID No. 19-28) in a bodily fluid is equal or below 100 ng / mL (100 ng / mL to 0 ng / mL), preferably equal or below 90 ng / mL (90 ng / mL to 0 ng / mL), more preferably equal or below 80 ng / mL (80 ng / mL to 0 ng / mL), more preferably equal or below 60 ng / mL (60 ng / mL to 0 ng / mL), more preferably equal or below 50 ng / mL (50 ng / mL to 0 ng / mL) and most preferably equal or below 40 ng / mL (40 ng / mL to 0 ng / mL) and / or the activity of PAM (SEQ ID No. 19-28) in a bodily fluid is equal or below 24 Units / L, more preferably equal or below 20 Units / L, more preferably equal or below 16 Units / L, more preferably equal or below 14 Units / L, and most preferably equal or below 10 Units / L and / or bio-ADM (SEQ ID No.: 5, 13-18) is equal or below 20 pg / mL (20 pg / mL-0 pg / mL), preferably equal or below 15 pg / mL (15 pg / mL 0 pg / mL), preferably equal or below 10 pg / mL (10 pg / mL-0 pg / mL), preferably equal or below 5 pg / mL (5 pg / mL - 0 pg / mL) and / or

[0155] ADM-Gly (SEQ ID No.: 4, 7-12) is equal or above 15 pg / mL, preferably equal or above 20 pg / mL, preferably equal or above 30 pg / mL, preferably is equal or above 50 pg / mL, together with the concentration of PAM (SEQ ID No. 19-28) in a bodily fluid is equal or below 100 ng / mL (100 ng / mL to 0 ng / mL), preferably equal or below 90 ng / mL (90 ng / mL to 0 ng / mL), more preferably equal or below 80 ng / mL (80 ng / mL to 0 ng / mL), more preferably equal or below 60 ng / mL (60 ng / mL to 0 ng / mL), more preferably equal or below 50 ng / mL (50 ng / mL to 0 ng / mL) and most preferably equal or below 40 ng / mL (40 ng / mL to 0 ng / mL).

[0156] An embodiment of the present invention is ADM-Gly (SEQ ID No.: 4, 7-12) for use in treatment of a disease in a subject, wherein the given levels of ADM-Gly may refer to a range of 15 10000 pg / mL, preferably to 20 8000 pg / mL, more preferably to 30- 5000 pg / mL, and most preferably in the range of 50-4500 pg / mL.

[0157] For disambiguation, the skilled person recognizes that embodiments of the present invention relate to ADM-Gly and / or a fragment thereof for use in the treatment of a disease in a subject, wherein the ratio between a) ADM-Gly and / or a fragment thereof (SEQ ID No.: 4, 7-12) and b) bio-ADM and / or a fragment thereof (SEQ ID No. 5, 13-18) in a bodily fluid of said subject before treatment is elevated over 1.5, preferably elevated over 1.6, more preferably elevated over 1.7, more preferably is elevated over 1.8, more preferable is elevated over 1.9, more preferably is higher than 2 and / or the concentration of PAM and / or an isoform of PAM and / or a fragment of PAM (SEQ ID No. 25-28 and / or 29-34) in a bodily fluid of said subject before treatment is equal or below 100 ng / mL, preferably equal or below 90 ng / mL, more preferably equal or below 80 ng / mL, more preferably equal or below 60 ng / mL, more preferably equal or below 50 ng / mL and most preferably equal or below 40 ng / mL and / or the activity of PAM and / or an isoform of PAM and / or a fragment of PAM (SEQ ID No. 25-28 and / or 29-34) in a bodily fluid of said subject before treatment is equal or below 24 Units / L, more preferably equal or below 20 Units / L, more preferably equal or below 16 Units / L, more preferably equal or below 14 Units / L, and most preferably equal or below 10 Units / L and / or bio-ADM and / or a fragment thereof (SEQ ID No.: 5, 13-18) in a bodily fluid of said subject before treatment is equal or below 20 pg / mL, preferably equal or below 15 pg / mL, preferably equal or below 10 pg / mL, preferably equal or below 5 pg / mL, and / or

[0158] ADM-Gly and / or a fragment thereof (SEQ ID No.: 4, 7-12) in a bodily fluid of said subject before treatment is equal or below 50 pg / mL, preferably equal or below 30 pg / mL, preferably equal or below 20 pg / mL, most preferred equal or below 15 pg / mL, and / or

[0159] ADM-Gly and / or a fragment thereof (SEQ ID No.: 4, 7-12) in a bodily fluid of said subject before treatment is equal or above 15 pg / mL, preferably equal or above 20 pg / mL, preferably equal or above 30 pg / mL, preferably is equal or above 50 pg / mL, together with the concentration of PAM and / or an isoform of PAM and / or a fragment of PAM (SEQ ID No. 25-28 and / or 29-34) in a bodily fluid of said subject before treatment is equal or below 100 ng / mL, preferably equal or below 90 ng / mL, more preferably equal or below 80 ng / mL, more preferably equal or below 60 ng / mL, more preferably equal or below 50 ng / mL and most preferably equal or below 40 ng / mL, and / or

[0160] ADM-Gly and / or a fragment thereof (SEQ ID No.: 4, 7-12) in a bodily fluid of said subject before treatment is equal or above 15 pg / mL, preferably equal or above 20 pg / mL, preferably above 30 pg / mL, preferably is equal or above 50 pg / mL, together with the activity of PAM and / or an isoform of PAM and / or a fragment of PAM (SEQ ID No. 25-28 and / or 29-34) in a bodily fluid is equal or below 24 Units / L, more preferably equal or below 20 Units / L, more preferably equal or below 16 Units / L, more preferably equal or below 14 Units / L and most preferably equal or below 10 Units / L.

[0161] Accordingly, the person skilled in the art is aware that further embodiments of the present invention relate to ADM-Gly and / or a fragment thereof for use in the treatment of a disease in a subject, wherein the ratio between a) ADM-Gly and / or a fragment thereof (SEQ ID No.: 4, 7-12) and b) bio-ADM and / or a fragment thereof (SEQ ID No. 5, 13-18) in a bodily fluid of said subject is monitored after said treatment and / or the concentration of PAM and / or an isoform of PAM and / or a fragment of PAM (SEQ ID No. 25-28 and / or 29-34) in a bodily fluid of said subject is monitored after said treatment and / or the activity of PAM and / or an isoform of PAM and / or a fragment of PAM (SEQ ID No. 25-28 and / or 29-34) in a bodily fluid of said subject is monitored after said treatment and / or bio-ADM and / or a fragment thereof (SEQ ID No.: 5, 13-18) in abodily fluid of said subject is monitored after said treatment and / or

[0162] ADM-Gly and / or a fragment thereof (SEQ ID No.: 4, 7-12) in a bodily fluid of said subject is monitored after said treatment and / or

[0163] ADM-Gly and / or a fragment thereof (SEQ ID No.: 4, 7-12) together with the concentration of PAM and / or an isoform of PAM and / or a fragment of PAM (SEQ ID No. 25-28 and / or 29-34) in a bodily fluid of said subject is monitored after said treatment, and / or

[0164] ADM-Gly and / or a fragment thereof (SEQ ID No.: 4, 7-12) together with the activity of PAM and / or an isoform of PAM and / or a fragment of PAM (SEQ ID No. 25-28 and / or 29-34) in a bodily fluid of said subject is monitored after said treatment.

[0165] Another embodiment of the present invention is ADM-Gly (SEQ ID No.: 4, 7-12) for use in treatment of a disease in a subject, wherein ADM-Gly (SEQ ID No.: 4, 7-12) is below 15 pg / mL (15 pg / mL - 0 pg / mL), preferably below 10 pg / mL (10 pg / mL - 0 pg / mL), preferably below 5 pg / mL (5 pg / mL - 0 pg / mL).

[0166] Subject matter according to the invention is ADM-Gly and fragments of thereof (SEQ ID No.: 4, 7-12) for use in treatment of a disease in a subject, wherein ADM-Gly is applied at a dosage of 0.1-500 pg / kg, preferably 1-400 pg / kg, more preferably 2-300 pg / mL, more preferably 3-200 pg / kg, even more preferably 5-100 pg / kg.

[0167] The skilled person readily recognizes that typically, pharmaceutical agents are administered to a subject in the unit weight / bodyweight of the subject. Subject matter according to the invention is therefore ADM-Gly and fragments of thereof (SEQ ID No.: 4, 7-12) for use in treatment of a disease in a subject, wherein ADM-Gly is applied at a dosage of 0.1-500 pg / kg, preferably 1-400 pg / kg, more preferably 2- 300 pg / kg, more preferably 3-200 pg / kg, even more preferably 5-100 pg / kg.

[0168] It follows that embodiments of the present invention refer to ADM-Gly and / or a fragment thereof for use in the treatment of a disease in a subject, wherein ADM-Gly is applied at a dosage of 0.1-500 pg / kg, preferably 1-400 pg / kg, more preferably 2-300 pg / mL, more preferably 2-300 pg / kg, more preferably 3-200 pg / kg, more preferably 5-100 pg / kg or wherein a fragment of ADM-Gly is applied at a corresponding molar dosage to said ADM-Gly.

[0169] In particular embodiments of the present invention, said pharmaceutical composition 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. orally)” likewise reads on “formulated for (e.g. oral) administration”.

[0170] In preferred embodiments of the present invention, said pharmaceutical composition 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. orally)” likewise reads on “formulated for (e.g. oral) administration. In the most preferred embodiments of the present invention, said pharmaceutical composition is formulated to be administered or is administered, subcutaneously, intradermally, intramuscularly, intraarterially or intravenously, or via intraperitoneal administration.

[0171] 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.

[0172] Subject matter of the present invention is ADM-Gly for use in treatment of a disease in a subject, wherein ADM-Gly is to be used alone or in combination 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.

[0173] An embodiment of the present invention is ADM-Gly for use in treatment of a disease in a subject, wherein ADM-Gly is in addition to be used with Vitamin C and wherein • 0.1-500 pg / kg of ADM-Gly is 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,

[0174] • more preferably 1-400 pg / kg of ADM-Gly is 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,

[0175] • more preferably 2-300 pg / kg of ADM-Gly is 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,

[0176] • more preferably 3-200 pg / kg ADM-Gly is 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,

[0177] • more preferably 5-100 pg / kg of ADM-Gly is 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, wherein 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 infusions, wherein one or all compounds are delivered as an injection and / or infusion or a distinct compound is injected and another distinct compound is infused. Said compounds or combinations thereof may be further administered using intramuscular or intraperitoneal or subcutaneous or intravenous application routes.

[0178] 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

[0179] Subject matter of the present invention is ADM-Gly and fragments thereof for use in treatment of a disease in a subject, wherein said ADM-Gly and fragments thereof are modified by pegylation, or with Albumins, or with Ig-Fc regions wherein Ig can be IgG, IgM or IgA, or is modified with protective groups, or ADM-Gly is modified with lipids, or ADM-Gly is modified via site-directed mutagenesis, or ADM-Gly is modified to function as a prodrug, or ADM-Gly is modified with natural or synthetic polymers.

[0180] Subject matter of the present invention is ADM-Gly and fragments thereof for use in treatment of a disease in a subject, wherein ADM-Gly and fragments thereof are modified by 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, more preferably modified to act as PEG-based carrier prodrug, 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.

[0181] Subject matter of the present invention is a method for a long-lasting elevation of amidated adrenomedullin in the circulation by the use of its glycine-extended ADM precursor alone and in one embodiment with the Vitamin C, as well as the use of the method to treat and / or prevent an acute and / or chronic disease with such compound.

[0182] The present invention encompasses a compound or a combination of compounds that is suitable for use in the pharmaceutical industry. This compound includes not only the compound itself, but also a pharmaceutically acceptable salt of the compound and a solvent that is considered safe for pharmaceutical use. It is worth noting that the pharmaceutically acceptable salt and solvate of the compound are not limited to any specific type, but the salt or solvate exemplified above is preferred. The compound, when in the form of a salt or solvate, can be used in various pharmaceutical applications. The compound shall mean an ADM-Gly and / or a combination of ADM-Gly with Vitamin C. Therefore, the compound of one aspect of the present invention is safe and has low toxicity. Accordingly, pharmaceuticals containing this embodiment of the present invention's compound or its pharmaceutically acceptable salts or solvates as active ingredients can be applied to various subjects requiring prevention or treatment of the symptoms, diseases, and / or disorders. Preferred subjects include human or non-human mammals (e.g. pigs, dogs, cows, rats, mice, guinea pigs, rabbits, chickens, sheep, cats, monkeys, orangutans, or chimpanzees), either as experimental subjects or patients.

[0183] By administering the pharmaceutical containing this embodiment of the present invention's compound to the subject, the symptoms, diseases, and / or disorders in the subject can be prevented or treated.

[0184] Throughout the specification the term “ADM-Gly” includes all splice variants of ADM-Gly if not stated otherwise.

[0185] 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.

[0186] Thus, the terms "comprising / including / having" mean that any further component (or likewise features, integers, steps and the like) can / may be present.

[0187] The term "consisting” of means that no further component (or likewise features, integers, steps and the like) is present.

[0188] 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.

[0189] The given definitions and explanations also apply mutatis mutandis to the following items. The present invention also relates to the following items:

[0190] 1. ADM-Gly for use in treatment of a disease in a subject, wherein ADM-Gly (SEQ ID No.:4, 7- 12) is in modified or unmodified form.

[0191] 2. ADM-Gly for use in treatment of a disease in a subject according to embodiment 1, wherein ADM-Gly is to be used in said subject in need of elevated level of bio-ADM (SEQ ID No.: 5, 13-18) in the circulation wherein elevated level means by at least 10 pg / mL, preferably by at least 20 pg / mL, more preferably by at least 30 pg / mL, more preferably by at least 40 pg / mL, most preferably by at least 50 pg / mL.

[0192] 3. ADM-Gly for use in treatment of a disease in a subject according to embodiments 1 or 2, wherein said disease is characterized by a ADM-Gly / bio-ADM ratio above a threshold in a sample of bodily fluid of said subject and / or a bio-ADM level below a certain threshold and / or a ADM-Gly level below a certain threshold and / or a ADM-Gly level above a certain threshold in combination with level of PAM and / or its isoforms and / or fragments thereof below a threshold and / or a level of P AM and / or its isoforms and / or fragments thereof below a threshold. 4. ADM-Gly for use in treatment of a disease in a subject according to any of embodiments 1 to 3, wherein ADM-Gly is to be used in treatment of the diseases, selected from the group comprising pulmonary hypertension and hypertension, type-2 diabetes, inflammatory bowel disease and / or Crohn’s disease, sepsis and / or septic shock, cerebral infarction, mild cognitive impairment (MCI) and Alzheimer’s Disease (AD).

[0193] 5. ADM-Gly for use in treatment of a disease in a subject according to any of embodiments 1 to 4, wherein ADM-Gly is to be used in combination with Vitamin C.

[0194] 6. ADM-Gly for use in treatment of a disease in a subject according to any of embodiments 1 to 5, wherein the ratio of ADM-Gly (SEQ ID No.: 4, 7-12) and bio-ADM (SEQ ID No.: 5, 13-18) (ADM- Gly / bio-ADM) in a bodily fluid is elevated over 1.5, preferably elevated over 1.6, more preferably elevated over 1.7, more preferably is elevated over 1.8, more preferable is elevated over 1.9, more preferably is higher than 2 and / or the concentration of PAM (SEQ ID No. 19-28) in a bodily fluid is equal or below 100 ng / mL, preferably equal or below 90 ng / mL, more preferably equal or below 80 ng / mL, more preferably equal or below 60 ng / mL, more preferably equal or below 50 ng / mL and most preferably equal or below 40 ng / mL and / or the activity of PAM (SEQ ID No. 19-28) in a bodily fluid is equal or below 24 Units / L, more preferably equal or below 20 Units / L, more preferably equal or below 16 Units / L, more preferably equal or below 14 Units / L, and most preferably equal or below 10 Units / L and / or bio-ADM (SEQ ID No.: 5, 13-18) is equal or below 20 pg / mL, preferably equal or below 15 pg / mL, preferably equal or below 10 pg / mL, preferably equal or below 5 pg / mL and / or

[0195] ADM-Gly (SEQ ID No.: 4, 7-12 ) is equal or above 15 pg / mL, preferably equal or above 20 pg / mL, preferably equal or above 30 pg / mL, preferably is equal or above 50 pg / mL, together with the concentration of PAM (SEQ ID No. 19-28) in a bodily fluid is equal or below 100 ng / mL, preferably equal or below 90 ng / mL, more preferably equal or below 80 ng / mL, more preferably equal or below 60 ng / mL, more preferably equal or below 50 ng / mL and most preferably equal or below 40 ng / mL.

[0196] 7. ADM-Gly (SEQ ID No.: 4, 7-12) is equal or above 15 pg / mL, preferably equal or above 20 pg / mL, preferably above 30 pg / mL, preferably is equal or above 50 pg / mL, together with the activity of PAM (SEQ ID No. 19-28) in a bodily fluid is equal or below 24 Units / L, more preferably equal or below 20 Units / L, more preferably equal or below 16 Units / L, more preferably equal or below 14 Units / L and most preferably equal or below 10 Units / L. ADM-Gly for use in treatment of a disease in a subject according to any of embodiments 1 to 6, wherein ADM-Gly is applied at a dosage of 0.1-500 pg / kg, preferably 1-400 pg / kg, more preferably 2-300 pg / mL, more preferably 2-300 pg / kg, more preferably 3-200 pg / kg, more preferably 5-100 pg / kg.

[0197] 8. ADM-Gly for use in treatment of a disease in a subject according to any of embodiments 1 to 7, wherein wherein ADM-Gly is to be used in combination 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.

[0198] 9. ADM-Gly for use in treatment of a disease in a subject according to any of embodiments 1 to 8, wherein said ADM-Gly is modified by 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, 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.

[0199] 1. The following embodiments form also part of the present inventiomA method of treating a disease in a subject, said method comprising administering to said subject an effective amount of ADM- Gly, wherein ADM-Gly (SEQ ID No.:4, 7-12) is to be used in modified or unmodified form.

[0200] 2. A method of treating a disease in a subject, said method comprising administering to said subject an effective amount of ADM-Gly according to claim 10, wherein ADM-Gly is to be used subject is in need of elevated level of bio- ADM in the circulation wherein elevated level means by at least 10 pg / mL, preferably by at least 20 pg / mL, more preferably by at least 30 pg / mL, more preferably by at least 40 pg / mL, most preferably by at least 50 pg / mL.

[0201] 3. A method of treating a disease in a subject, said method comprising administering to said subject an effective amount of ADM-Gly according to claim 10 or 11, wherein said disease is characterized by an ADM-Gly / bio-ADM ratio above a threshold in a sample of bodily fluid of said subject and / or a bio-ADM level below a certain threshold and / or

[0202] ADM-Gly level below a certain threshold an ADM-Gly level above a certain threshold in combination with level of PAM and / or its isoforms and / or fragments thereof below a threshold and / or a level of PAM and / or its isoforms and / or fragments thereof below a threshold. A method of treating a disease in a subject, said method comprising administering to said subject an effective amount of ADM-Gly according to any of claims 10 to 12, wherein said disease is selected from the group comprising pulmonary hypertension and hypertension, type-2 diabetes, inflammatory bowel disease and / or Crohn's disease, sepsis and / or septic shock, cerebral infarction, mild cognitive impairment (MCI) and Alzheimer’s Disease (AD). A method of treating a disease in a subject, said method comprising administering to said subject an effective amount of ADM-Gly according to any of claims 10 to 13, wherein ADM-Gly is to be used in combination with Vitamin C. A method of treating a disease in a subject, said method comprising administering to said subject an effective amount of ADM-Gly according to any of claims 10 to 14, wherein the ratio of ADM-Gly (SEQ ID No.: 4, 7-12) and bio-ADM (SEQ ID No.: 5, 13-18) (ADM-Gly / bio- ADM) in a bodily fluid is elevated over 1.5, preferably elevated over 1.6, more preferably elevated over 1.7, more preferably is elevated over 1.8, more preferable is elevated over 1.9, more preferably is higher than 2 and / or the concentration of PAM (SEQ ID No. 19-28) in a bodily fluid is equal or below 100 ng / mL, preferably equal or below 90 ng / mL, more preferably equal or below 80 ng / mL, more preferably equal or below 60 ng / mL, more preferably equal or below 50 ng / mL and most preferably equal or below 40 ng / mL and / or the activity of PAM (SEQ ID No. 19-28) in a bodily fluid is equal or below 24 Units / L, more preferably equal or below 20 Units / L, more preferably equal or below 16 Units / L, more preferably equal or below 14 Units / L, specified in example 2, and most preferably equal or below 10 Units / L and / or bio-ADM (SEQ ID No.: 5, 13-18) is equal or below 20 pg / mL, preferably equal or below 15 pg / mL, preferably equal or below 10 pg / mL, preferably equal or below 5 pg / mL and / or

[0203] ADM-Gly (SEQ ID No.: 4, 7-12) is equal or above 15 pg / mL, preferably equal or above 20 pg / mL, preferably equal or above 30 pg / mL, preferably is equal or above 50 pg / mL, together with the concentration of PAM (SEQ ID No. 19-28) in a bodily fluid is equal or below 100 ng / mL, preferably equal or below 90 ng / mL, more preferably equal or below 80 ng / mL, more preferably equal or below 60 ng / mL, more preferably equal or below 50 ng / mL and most preferably equal or below 40 ng / mL and / or ADM-Gly (SEQ ID No.: 4, 7-12) is equal or above 15 pg / mL, preferably equal or above 20 pg / mL, preferably equal or above 30 pg / mL, preferably is equal or above 50 pg / mL, together with the activity of PAM (SEQ ID No. 19-28) in a bodily fluid is equal or below 24 Units / L, more preferably equal or below 20 Units / L, more preferably equal or below 16 Units / L, more preferably equal or below 14 Units / L and most preferably equal or below 10 Units / L.

[0204] 7. A method of treating a disease in a subject, said method comprising administering to said subject an effective amount of ADM-Gly according to any of claims 10 to 15, wherein ADM-Gly is applied at a dosage of 0.1-500 pg / kg, preferably 1-400 pg / kg, more preferably 2-300 pg / mL, more preferably 2-300 pg / kg, more preferably 3-200 pg / kg, more preferably 5-100 pg / kg.

[0205] 8. A method of treating a disease in a subject, said method comprising administering to said subject an effective amount of ADM-Gly according to any of claims 10 to 16, wherein ADM-Gly is to be used in combination 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.

[0206] 9. A method of treating a disease in a subject, said method comprising administering to said subject an effective amount of ADM-Gly according to any of claims 10 to 17 wherein ADM-Gly is modified by 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, 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.

[0207] For disambiguation: The following items form also part of the present invention

[0208] Aspect A:

[0209] The given definitions and explanations also apply mutatis mutandis to the following items. The present invention also relates to the following items:

[0210] 1. ADM-Gly for use in treatment of a disease in a subject, wherein ADM-Gly (SEQ ID No.:4, 7-

[0211] 12) is in modified or unmodified form. 2. ADM-Gly for use in treatment of a disease in a subject according to item 1 of aspect A, wherein ADM-Gly is to be used in said subject in need of elevated level of bio-ADM (SEQ ID No.: 5, 13-18) in the circulation wherein elevated level means by at least 10 pg / mL, preferably by at least 20 pg / mL, more preferably by at least 30 pg / mL, more preferably by at least 40 pg / mL, most preferably by at least 50 pg / mL.

[0212] 3. ADM-Gly for use in treatment of a disease in a subject according to items 1 or 2 of aspect A, wherein said disease is characterized by a ADM-Gly / bio-ADM ratio above a threshold in a sample of bodily fluid of said subject and / or a bio-ADM level below a certain threshold and / or a ADM-Gly level below a certain threshold and / or a ADM-Gly level above a certain threshold in combination with level of PAM and / or its isoforms and / or fragments thereof below a threshold and / or a level of PAM and / or its isoforms and / or fragments thereof below a threshold.

[0213] 4. ADM-Gly for use in treatment of a disease in a subject according to any of items 1 to 3 of aspect A, wherein ADM-Gly is to be used in treatment of the diseases, selected from the group comprising pulmonary hypertension and hypertension, type-2 diabetes, inflammatory bowel disease and / or Crohn’s disease, sepsis and / or septic shock, cerebral infarction, mild cognitive impairment (MCI) and Alzheimer’s Disease (AD).

[0214] 5. ADM-Gly for use in treatment of a disease in a subject according to any of items 1 to 4 of aspect A, wherein ADM-Gly is to be used in combination with Vitamin C.

[0215] 6. ADM-Gly for use in treatment of a disease in a subject according to any of items 1 to 5 of aspect A, wherein the ratio of ADM-Gly (SEQ ID No.: 4, 7-12) and bio-ADM (SEQ ID No.: 5, 13-18) (ADM- Gly / bio-ADM) in a bodily fluid is elevated over 1.5, preferably elevated over 1.6, more preferably elevated over 1.7, more preferably is elevated over 1.8, more preferable is elevated over 1.9, more preferably is higher than 2 and / or the concentration of PAM (SEQ ID No. 19-28) in a bodily fluid is equal or below 100 ng / mL, preferably equal or below 90 ng / mL, more preferably equal or below 80 ng / mL, more preferably equal or below 60 ng / mL, more preferably equal or below 50 ng / mL and most preferably equal or below 40 ng / mL and / or the activity of PAM (SEQ ID No. 19-28) in a bodily fluid is equal or below 24 Units / L, more preferably equal or below 20 Units / L, more preferably equal or below 16 Units / L, more preferably equal or below 14 Units / L, and most preferably equal or below 10 Units / L and / or bio-ADM (SEQ ID No.: 5, 13-18) is equal or below 20 pg / mL, preferably equal or below 15 pg / mL, preferably equal or below 10 pg / mL, preferably equal or below 5 pg / mL and / or

[0216] ADM-Gly (SEQ ID No.: 4, 7-12 ) is equal or above 15 pg / mL, preferably equal or above 20 pg / mL, preferably equal or above 30 pg / mL, preferably is equal or above 50 pg / mL, together with the concentration of P AM (SEQ ID No. 19-28) in a bodily fluid is equal or below 100 ng / mL, preferably equal or below 90 ng / mL, more preferably equal or below 80 ng / mL, more preferably equal or below 60 ng / mL, more preferably equal or below 50 ng / mL and most preferably equal or below 40 ng / mL and / or

[0217] ADM-Gly (SEQ ID No.: 4, 7-12) is equal or above 15 pg / mL, preferably equal or above 20 pg / mL, preferably above 30 pg / mL, preferably is equal or above 50 pg / mL, together with the activity of PAM (SEQ ID No. 19-28) in a bodily fluid is equal or below 24 Units / L, more preferably equal or below 20 Units / L, more preferably equal or below 16 Units / L, more preferably equal or below 14 Units / L and most preferably equal or below 10 Units / L.

[0218] 7. ADM-Gly for use in treatment of a disease in a subject according to any of items 1 to 6 of aspect A, wherein ADM-Gly is applied at a dosage of 0.1-500 pg / kg, preferably 1-400 pg / kg, more preferably 2- 300 pg / mL, more preferably 2-300 pg / kg, more preferably 3-200 pg / kg, more preferably 5-100 pg / kg.

[0219] 8. ADM-Gly for use in treatment of a disease in a subject according to any of items 1 to 7 of aspect A, wherein ADM-Gly is to be used in combination 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.

[0220] 9. ADM-Gly for use in treatment of a disease in a subject according to any of items 1 to 8 of aspect A, wherein said ADM-Gly is modified by 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, 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. Aspect B:

[0221] 1. A method of treating a disease in a subject, said method comprising administering to said subject an effective amount of ADM-Gly, wherein ADM-Gly (SEQ ID No.: 4, 7-12) is to be used in modified or unmodified form.

[0222] 2. A method of treating a disease in a subject, said method comprising administering to said subject an effective amount of ADM-Gly according to item 1 of aspect B, wherein ADM-Gly is to be used subject is in need of elevated level of bio-ADM in the circulation wherein elevated level means by at least 10 pg / mL, preferably by at least 20 pg / mL, more preferably by at least 30 pg / mL, more preferably by at least 40 pg / mL, most preferably by at least 50 pg / mL.

[0223] 3. A method of treating a disease in a subject, said method comprising administering to said subject an effective amount of ADM-Gly according to items 1 or 2 of aspect B, wherein said disease is characterized by an ADM-Gly / bio-ADM ratio above a threshold in a sample of bodily fluid of said subject and / or an bio-ADM level below a certain threshold and / or

[0224] ADM-Gly level below a certain threshold an ADM-Gly level above a certain threshold in combination with level of PAM and / or its isoforms and / or fragments thereof below a threshold and / or a level of PAM and / or its isoforms and / or fragments thereof below a threshold.

[0225] 4. A method of treating a disease in a subject, said method comprising administering to said subject an effective amount of ADM-Gly according to any of items 1 to 3 of aspect B, wherein said disease is selected from the group comprising pulmonary hypertension and hypertension, type-2 diabetes, inflammatory bowel disease and / or Crohn's disease, sepsis and / or septic shock, cerebral infarction, mild cognitive impairment (MCI) and Alzheimer’s Disease (AD).

[0226] 5. A method of treating a disease in a subject, said method comprising administering to said subject an effective amount of ADM-Gly according to any of items 1 to 4 of aspect B, wherein ADM-Gly is to be used in combination with Vitamin C.

[0227] 6. A method of treating a disease in a subject, said method comprising administering to said subject an effective amount of ADM-Gly according to any of items 1 to 5 of aspect B, wherein the ratio of ADM-Gly (SEQ ID No.: 4, 7-12) and bio-ADM (SEQ ID No.: 5, 13-18) (ADM-Gly / bio- ADM) in a bodily fluid is elevated over 1.5, preferably elevated over 1.6, more preferably elevated over 1.7, more preferably is elevated over 1.8, more preferable is elevated over 1.9, more preferably is higher than 2 and / or the concentration of PAM (SEQ ID No. 25-28 and / or 29-34) in a bodily fluid is equal or below 100 ng / mL, preferably equal or below 90 ng / mL, more preferably equal or below 80 ng / mL, more preferably equal or below 60 ng / mL, more preferably equal or below 50 ng / mL and most preferably equal or below 40 ng / mL and / or the activity of PAM (SEQ ID No. 25-28 and / or 29-34) in a bodily fluid is equal or below 24 Units / L, more preferably equal or below 20 Units / L, more preferably equal or below 16 Units / L, more preferably equal or below 14 Units / L, specified in example 2, and most preferably equal or below 10 Units / L and / or bio-ADM (SEQ ID No.: 5, 13-18) is equal or below 20 pg / mL, preferably equal or below 15 pg / mL, preferably equal or below 10 pg / mL, preferably equal or below 5 pg / mL and / or

[0228] ADM-Gly (SEQ ID No.: 4, 7-12) is equal or above 15 pg / mL, preferably equal or above 20 pg / mL, preferably equal or above 30 pg / mL, preferably is equal or above 50 pg / mL, together with the concentration of PAM (SEQ ID No. 25-28 and / or 29-34) in a bodily fluid is equal or below 100 ng / mL, preferably equal or below 90 ng / mL, more preferably equal or below 80 ng / mL, more preferably equal or below 60 ng / mL, more preferably equal or below 50 ng / mL and most preferably equal or below 40 ng / mL and / or

[0229] ADM-Gly (SEQ ID No.: 4, 7-12) is equal or above 15 pg / mL, preferably equal or above 20 pg / mL, preferably equal or above 30 pg / mL, preferably is equal or above 50 pg / mL, together with the activity of PAM (SEQ ID No. 25-28 and / or 29-34) in a bodily fluid is equal or below 24 Units / L, more preferably equal or below 20 Units / L, more preferably equal or below 16 Units / L, more preferably equal or below 14 Units / L and most preferably equal or below 10 Units / L. A method of treating a disease in a subject, said method comprising administering to said subject an effective amount of ADM-Gly according to any of items 1 to 6 of aspect B, wherein ADM-Gly is applied at a dosage of 0.1-500 pg / kg, preferably 1-400 pg / kg, more preferably 2-300 pg / mL, more preferably 2-300 pg / kg, more preferably 3-200 pg / kg, more preferably 5-100 pg / kg. 8. A method of treating a disease in a subject, said method comprising administering to said subject an effective amount of ADM-Gly according to any of items 1 to 7 of aspect B, wherein ADM-Gly is to be used in combination 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.

[0230] 9. A method of treating a disease in a subject, said method comprising administering to said subject an effective amount of ADM-Gly according to any of items 1 to 8 of aspect B wherein ADM-Gly is modified by 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, 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.

[0231] The present invention is further described by reference to the following non-limiting figures.

[0232] Figure Description

[0233] Fig. 1: The product of the Adrenomedullin gene, the Proadrenomedullin encodes for four peptide hormones and undergoes several enzymatic cleavage steps mainly by prohormone convertases (PCs) to release said peptide hormones. PAMP-Gly and ADM-Gly are inactive and can be activated via amidation into their active forms, PAMP-NH2 and bio-ADM respectively.

[0234] Fig. 2A: Representative calibration curve of the ADM-Gly Assay with synthetic human 1-53 ADM-Gly

[0235] Fig. 2B: Frequency distribution (histogram) of ADM-Gly (pg / mL) in self-reported healthy individuals (n=157)

[0236] Fig. 3A: Representative calibration curve of PAM Amidating Activity Assay (PAM-AMA).

[0237] Fig. 3B: Frequency distribution (histogram) of PAM-AMA in self-reported healthy individuals (n=120)

[0238] Fig. 3C: Correlation of PAM-AMA in matrix duplets (Li-heparin and serum) from self-reported healthy individuals (n=20)

[0239] Fig. 4A: Typical calibration curves of PAM sandwich immunoassay with recombinant PAM as calibration material in set-up utilizing antibodies against conformational peptides. Solid phase: antibody directed against PAL subunit, tracer: antibody directed against PHM subunit. Fig. 4B: Frequency distribution (histogram) of PAM concentration (conformational PAM-LIA) in healthy individuals (n=4106).

[0240] Fig. 5: Bio-ADM levels in rats before (=BL) and after the infusion of ADM-Gly (black bars), ADM- Gly in combination with Vitamin C (open bars).

[0241] Fig. 6: Determination of human 1-53 ADM-Gly half-life in circulation of rats. The half-life of ADM- Gly of 25.06 minutes was determined with the exponential one-phase decay algorithm of GraphPad Prism software Version 7.0.

[0242] Fig. 7: Effect of an intraperitoneal treatment with ADM-Gly in a murine CLP model 24 hours before CLP model induction on blood brain barrier integrity. Effect of treatment on blood-brain barrier integrity was measured as Evans-Blue (EB) extravasation in brain tissue homogenates in pg EB per g brain homogenate tissue. Right side: Exemplary brains are shown for the SHAM and CLP arms of the study.

[0243] SEQUENCES

[0244] SEQ ID No: 1 - pre-proADM (aa 1-185)

[0245] 10 20 30 40 50

[0246] MKLVSVALMY LGSLAFLGAD TARLDVASEF RKKWNKWALS RGKRELRMSS

[0247] 60 70 80 90 100

[0248] SYPTGLADVK AGPAQTLIRP QDMKGASRSP EDSSPDAARI RVKRYRQSMN

[0249] 110 120 130 140 150

[0250] NFQGLRSFGC RFGTCTVQKL AHQIYQFTDK DKDNVAPRSK ISPQGYGRRR 160 170 180

[0251] RRSLPEAGPG RTLVSSKPQA HGAPAPPSGS APHFL

[0252] SEQ ID No: 2 - PAMP-Gly (aa 22-42 of pre-proADM SEQ ID No. 11)

[0253] 10 20 30 40 50

[0254] ARLDVASEFR KKWNKWALSR G

[0255] SEQ ID No: 3 - MR-proADM (aa 45-92 of pre-proADM SEQ ID No. 11)

[0256] 10 20 30 40 50

[0257] ELRMSSSYPT GLADVKAGPA QTLIRPQDMK GASRSPEDSS PDAARTRV

[0258] SEQ ID No: 4 - ADM-Gly (aa 95-147 of pre-proADM SEQ ID No. 11)

[0259] 10 20 30 40 50

[0260] YRQSMNNFQG LRSFGCRFGT CTVQKLAHQI YQFTDKDKDN VAPRSKISPQ GYG

[0261] SEQ ID No: 5 - bio-ADM (aa 95-146 of pre-proADM SEQ ID No. 11)

[0262] 10 20 30 40 50

[0263] YRQSMNNFQG LRSFGCRFGT CTVQKLAHQI YQFTDKDKDN VAPRSKI SPQ GY SEQ ID No: 6 - CT-proADM (aa 148-185 of pre-proADM SEQ ID No. 11)

[0264] 10 20 30 40 50

[0265] RRRRRSLPEA GPGRTLVSSK PQAHGAPAPP SGSAPHFL

[0266] SEQ ID No: 7 - ADM-Gly 2-53 (aa 96-147 of pre-proADM SEQ ID No. 11)

[0267] 10 20 30 40 50

[0268] RQSMNNFQG LRSFGCRFGT CTVQKLAHQI YQFTDKDKDN VAPRSKISPQ GYG

[0269] SEQ ID No: 8 - ADM-Gly 8-53 (aa 102-147 of pre-proADM SEQ ID No. 11)

[0270] 10 20 30 40 50

[0271] FQG LRSFGCRFGT CTVQKLAHQI YQFTDKDKDN VAPRSKISPQ GYG

[0272] SEQ ID No: 9 - ADM-Gly 22-53 (aa 115-147 of pre-proADM SEQ ID No. 11)

[0273] 10 20 30 40 50

[0274] TVQKLAHQI YQFTDKDKDN VAPRSKISPQ GYG

[0275] SEQ ID No: 10 - ADM-Gly 27-53 (aa 120-147 of pre-proADM SEQ ID No. 11)

[0276] 10 20 30 40 50

[0277] AHQI YQFTDKDKDN VAPRSKISPQ GYG

[0278] SEQ ID No: 11 - ADM-Gly 33-53 (aa 126-147 of pre-proADM SEQ ID No. 11)

[0279] 10 20 30 40 50

[0280] FTDKDKDN VAPRSKISPQ GYG

[0281] SEQ ID No: 12 - ADM-Gly 49-53 (aa 143-147 of pre-proADM SEQ ID No. 11)

[0282] 10 20 30 40 50

[0283] PQGYG

[0284] SEQ ID No: 13 - bio-ADM 2-52 (aa 96-146 of pre-proADM SEQ ID No. 11)

[0285] 10 20 30 40 50

[0286] RQSMNNFQG LRSFGCRFGT CTVQKLAHQI YQFTDKDKDN VAPRSKISPQ GY

[0287] SEQ ID No: 14 - bio-ADM 8-52 (aa 102-146 of pre-proADM SEQ ID No. 11)

[0288] 10 20 30 40 50

[0289] FQG LRSFGCRFGT CTVQKLAHQI YQFTDKDKDN VAPRSKISPQ GY

[0290] SEQ ID No: 15 - bio-ADM 22-52 (aa 115-146 of pre-proADM SEQ ID No. 11)

[0291] 10 20 30 40 50

[0292] TVQKLAHQI YQFTDKDKDN VAPRSKISPQ GY

[0293] SEQ ID No: 16 - bio-ADM 27-52 (aa 120-146 of pre-proADM SEQ ID No. 11)

[0294] 10 20 30 40 50

[0295] AHQI YQFTDKDKDN VAPRSKISPQ GY SEQ ID No: 17 - bio-ADM 33-52 (aa 126-146 of pre-proADM SEQ ID No. 11)

[0296] 10 20 30 40 50

[0297] FTDKDKDN VAPRSKI SPQ GY

[0298] SEQ ID No: 18 - bio-ADM 49-52 (aa 143-146 of pre-proADM SEQ ID No. 11)

[0299] 10 20 30 40 50

[0300] PQGY

[0301] SEQ ID NO: 19 - Prepro-PAM isoform 1 AS 1-973

[0302] 10 20 30 40 50

[0303] MAGRVPSLLV LLVFPSSCLA FRS PLSVFKR FKETTRPFSN ECLGTTRPW

[0304] 60 70 80 90 100

[0305] PI DS SDFALD IRMPGVTPKQ SDTYFCMSMR I PVDEEAFVI DFKPRASMDT 110 120 130 140 150

[0306] VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA NILYAWARNA PPTRLPKGVG 160 170 180 190 200

[0307] FRVGGETGSK YFVLQVHYGD I SAFRDNNKD CSGVSLHLTR LPQPLIAGMY 210 220 230 240 250

[0308] LMMSVDTVI P AGEKWNSDI SCHYKNYPMH VFAYRVHTHH LGKVVSGYRV 260 270 280 290 300

[0309] RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF GDLLAARCVF TGEGRTEATH 310 320 330 340 350

[0310] IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT QNVAPDMFRT I PPEANI PI P 360 370 380 390 400

[0311] VKSDMVMMHE HHKETEYKDK I PLLQQPKRE EEEVLDQGDF YSLLSKLLGE 410 420 430 440 450

[0312] REDVVHVHKY NPTEKAESES DLVAE IANVV QKKDLGRSDA REGAEHERGN 460 470 480 490 500

[0313] AILVRDRIHK FHRLVSTLRP PESRVFSLQQ PPPGEGTWEP EHTGDFHMEE 510 520 530 540 550

[0314] ALDWPGVYLL PGQVSGVALD PKNNLVIFHR GDHVWDGNSF DSKFVYQQIG 560 570 580 590 600

[0315] LGPIEEDT IL VI DPNNAAVL QSSGKNLFYL PHGLS IDKDG NYWVTDVALH 610 620 630 640 650

[0316] QVFKLDPNNK EGPVLILGRS MQPGSDQNHF CQPTDVAVDP GTGAIYVSDG

[0317] 660 670 680 690 700

[0318] YCNSRIVQFS PSGKFI TQWG EES SGSSPLP GQFTVPHSLA LVPLLGQLCV 710 720 730 740 750

[0319] ADRENGRIQC FKTDTKEFVR E IKHS SFGRN VFAI SYI PGL LFAVNGKPHF 7 60 770 780 790 800

[0320] GDQEPVQGFV MNFSNGE I ID I FKPVRKHFD MPHDIVASED GTVYIGDAHT 810 820 830 840 850

[0321] NTVWKFTLTE KLEHRSVKKA GIEVQEIKEA EAVVETKMEN KPTS SELQKM 860 870 880 890 900

[0322] QEKQKLIKEP GSGVPVVLIT TLLVI PVWL LAIAI FIRWK KSRAFGDSEH 910 920 930 940 950

[0323] KLETSSGRVL GRFRGKGSGG LNLGNFFASR KGYSRKGFDR LSTEGSDQEK 960 970

[0324] EDDGSESEEE YSAPLPALAP SSS

[0325] SEQ ID NO: 20 - Prepro-PAM isoform 2 AS 1-868

[0326] 10 20 30 40 50

[0327] MAGRVPSLLV LLVFPS SCLA FRSPLSVFKR FKETTRPFSN ECLGTTRPVV 60 70 80 90 100

[0328] PI DS SDFALD IRMPGVTPKQ SDTYFCMSMR I PVDEEAFVI DFKPRASMDT 110 120 130 140 150

[0329] VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA NILYAWARNA PPTRLPKGVG 160 170 180 190 200

[0330] FRVGGETGSK YFVLQVHYGD I SAFRDNNKD CSGVSLHLTR LPQPLIAGMY 210 220 230 240 250

[0331] LMMSVDTVI P AGEKWNSDI SCHYKNYPMH VFAYRVHTHH LGKVVSGYRV

[0332] 260 270 280 290 300

[0333] RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF GDLLAARCVF TGEGRTEATH 310 320 330 340 350

[0334] IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT QNVAPDMFRT I PPEANI PI P 360 370 380 390 400

[0335] VKSDMVMMHE HHKETEYKDK I PLLQQPKRE EEEVLDQDFH MEEALDWPGV 410 420 430 440 450

[0336] YLLPGQVSGV ALDPKNNLVI FHRGDHVWDG NSFDSKFVYQ QIGLGPIEED 460 470 480 490 500

[0337] TILVI DPNNA AVLQSSGKNL FYLPHGLS ID KDGNYWVTDV ALHQVFKLDP 510 520 530 540 550

[0338] NNKEGPVLIL GRSMQPGSDQ NHFCQPTDVA VDPGTGAIYV SDGYCNSRIV 560 570 580 590 600

[0339] QFSPSGKFIT QWGEESSGSS PLPGQFTVPH SLALVPLLGQ LCVADRENGR 610 620 630 640 650

[0340] IQCFKTDTKE FVREIKHS SF GRNVFAI SYI PGLLFAVNGK PHFGDQEPVQ

[0341] 660 670 680 690 700

[0342] GFVMNFSNGE I I DI FKPVRK HFDMPHDIVA SEDGTVYIGD AHTNTVWKFT 710 720 730 740 750

[0343] LTEKLEHRSV KKAGIEVQE I KEAEAWETK MENKPTSSEL QKMQEKQKLI 7 60 770 780 790 800

[0344] KEPGSGVPVV LITTLLVI PV VVLLAIAI FI RWKKSRAFGD SEHKLETSSG 810 820 830 840 850

[0345] RVLGRFRGKG SGGLNLGNFF ASRKGYSRKG FDRLSTEGSD QEKEDDGSES 860

[0346] EEEYSAPLPA LAPSSS

[0347] SEQ ID No: 21 - Prepro-PAM isoform 3 AS (amino acids 829-896 of SEQ ID No. 1 missing)

[0348] 10 20 30 40 50

[0349] MAGRVPSLLV LLVFPSSCLA FRSPLSVFKR FKETTRPFSN ECLGTTRPVV 60 70 80 90 100

[0350] PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR I PVDEEAFVI DFKPRASMDT 110 120 130 140 150

[0351] VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA NILYAWARNA PPTRLPKGVG 160 170 180 190 200

[0352] FRVGGETGSK YFVLQVHYGD I SAFRDNNKD CSGVSLHLTR LPQPLIAGMY 210 220 230 240 250

[0353] LMMSVDTVI P AGEKVVNSDI SCHYKNYPMH VFAYRVHTHH LGKWSGYRV

[0354] 260 270 280 2 90 300

[0355] RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF GDLLAARCVF TGEGRTEATH

[0356] 310 320 330 340 350

[0357] IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT QNVAPDMFRT I PPEANI PI P

[0358] 360 370 380 390 400

[0359] VKSDMVMMHE HHKETEYKDK I PLLQQPKRE EEEVLDQGDF YSLLSKLLGE 410 420 430 440 450

[0360] REDVVHVHKY NPTEKAESES DLVAEIANVV QKKDLGRSDA REGAEHERGN

[0361] 460 470 480 4 90 500

[0362] AILVRDRIHK FHRLVSTLRP PESRVFSLQQ PPPGEGTWEP EHTGDFHMEE

[0363] 510 520 530 540 550

[0364] ALDWPGVYLL PGQVSGVALD PKNNLVI FHR GDHVWDGNSF DSKFVYQQIG

[0365] 560 570 580 590 600

[0366] LGPIEEDT IL VI DPNNAAVL QSSGKNLFYL PHGLS I DKDG NYWVTDVALH

[0367] 610 620 630 640 650

[0368] QVFKLDPNNK EGPVLILGRS MQPGSDQNHF CQPTDVAVDP GTGAIYVSDG

[0369] 660 670 680 690 700

[0370] YCNSRIVQFS PSGKFITQWG EESSGSSPLP GQFTVPHSLA LVPLLGQLCV

[0371] 710 720 730 740 750

[0372] ADRENGRIQC FKTDTKEFVR EIKHSSFGRN VFAI SYI PGL LFAVNGKPHF

[0373] 7 60 770 780 7 90 800

[0374] GDQEPVQGFV MNFSNGE I ID I FKPVRKHFD MPHDIVASED GTVYIGDAHT

[0375] 810 820 830 840 850

[0376] NTVWKFTLTE KLEHRSVKKA GIEVQE IKDS EHKLETSSGR VLGRFRGKGS

[0377] 860 870 880 890 900

[0378] GGLNLGNFFA SRKGYSRKGF DRLSTEGSDQ EKEDDGSESE EEYSAPLPAL 905

[0379] APS S S

[0380] SEQ ID No: 22 - Prepro-PAM isoform 4 (amino acids 829-914 of SEQ ID No. 1 missing)

[0381] 10 20 30 40 50

[0382] MAGRVPSLLV LLVFPSSCLA FRSPLSVFKR FKETTRPFSN ECLGTTRPVV

[0383] 60 70 80 90 100

[0384] PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR I PVDEEAFVI DFKPRASMDT 110 120 130 140 150

[0385] VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA NILYAWARNA PPTRLPKGVG 160 170 180 190 200

[0386] FRVGGETGSK YFVLQVHYGD I SAFRDNNKD CSGVSLHLTR LPQPLIAGMY 210 220 230 240 250

[0387] LMMSVDTVI P AGEKVVNSDI SCHYKNYPMH VFAYRVHTHH LGKWSGYRV 260 270 280 2 90 300

[0388] RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF GDLLAARCVF TGEGRTEATH 310 320 330 340 350

[0389] IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT QNVAPDMFRT I PPEANI PI P 360 370 380 390 400 VKSDMVMMHE HHKETEYKDK I PLLQQPKRE EEEVLDQGDF YSLLSKLLGE 410 420 430 440 450

[0390] REDVVHVHKY NPTEKAESES DLVAEIANVV QKKDLGRSDA REGAEHERGN 460 470 480 4 90 500

[0391] AILVRDRIHK FHRLVSTLRP PESRVFSLQQ PPPGEGTWEP EHTGDFHMEE 510 520 530 540 550

[0392] ALDWPGVYLL PGQVSGVALD PKNNLVI FHR GDHVWDGNSF DSKFVYQQIG 560 570 580 590 600

[0393] LGPIEEDT IL VI DPNNAAVL QSSGKNLFYL PHGLS I DKDG NYWVTDVALH 610 620 630 640 650

[0394] QVFKLDPNNK EGPVLILGRS MQPGSDQNHF CQPTDVAVDP GTGAIYVSDG 660 670 680 690 700

[0395] YCNSRIVQFS PSGKFITQWG EESSGSSPLP GQFTVPHSLA LVPLLGQLCV 710 720 730 740 750

[0396] ADRENGRIQC FKTDTKEFVR EIKHSSFGRN VFAI SYI PGL LFAVNGKPHF 7 60 770 780 7 90 800

[0397] GDQEPVQGFV MNFSNGE I ID I FKPVRKHFD MPHDIVASED GTVYIGDAHT 810 820 830 840 850

[0398] NTVWKFTLTE KLEHRSVKKA GIEVQE IKGK GSGGLNLGNF FASRKGYSRK 860 870 880

[0399] GFDRLSTEGS DQEKEDDGSE SEEEYSAPLP ALAPSSS

[0400] SEQ ID No: 23 - Prepro-PAM Isoform 5 (Isoform 1 with an additional aa in position 896)

[0401] 10 20 30 40 50

[0402] MAGRVPSLLV LLVFPSSCLA FRSPLSVFKR FKETTRPFSN ECLGTTRPVV 60 70 80 90 100

[0403] PI DS SDFALD IRMPGVTPKQ SDTYFCMSMR I PVDEEAFVI DFKPRASMDT 110 120 130 140 150

[0404] VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA NILYAWARNA PPTRLPKGVG 160 170 180 190 200

[0405] FRVGGETGSK YFVLQVHYGD I SAFRDNNKD CSGVSLHLTR LPQPLIAGMY 210 220 230 240 250

[0406] LMMSVDTVI P AGEKVVNSDI SCHYKNYPMH VFAYRVHTHH LGKWSGYRV 260 270 280 2 90 300

[0407] RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF GDLLAARCVF TGEGRTEATH 310 320 330 340 350

[0408] IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT QNVAPDMFRT I PPEANI PI P 360 370 380 390 400

[0409] VKSDMVMMHE HHKETEYKDK I PLLQQPKRE EEEVLDQGDF YSLLSKLLGE 410 420 430 440 450

[0410] REDVVHVHKY NPTEKAESES DLVAEIANVV QKKDLGRSDA REGAEHERGN 460 470 480 4 90 500

[0411] AILVRDRIHK FHRLVSTLRP PESRVFSLQQ PPPGEGTWEP EHTGDFHMEE 510 520 530 540 550

[0412] ALDWPGVYLL PGQVSGVALD PKNNLVI FHR GDHVWDGNSF DSKFVYQQIG 560 570 580 590 600

[0413] LGPIEEDT IL VI DPNNAAVL QSSGKNLFYL PHGLS I DKDG NYWVTDVALH 610 620 630 640 650

[0414] QVFKLDPNNK EGPVLILGRS MQPGSDQNHF CQPTDVAVDP GTGAIYVSDG 660 670 680 690 700

[0415] YCNSRIVQFS PSGKFITQWG EESSGSSPLP GQFTVPHSLA LVPLLGQLCV 710 720 730 740 750

[0416] ADRENGRIQC FKTDTKEFVR EIKHSSFGRN VFAI SYI PGL LFAVNGKPHF 7 60 770 780 7 90 800

[0417] GDQEPVQGFV MNFSNGE I ID I FKPVRKHFD MPHDIVASED GTVYIGDAHT

[0418] 810 820 830 840 850

[0419] NTVWKFTLTE KLEHRSVKKA GIEVQE IKEA EAWETKMEN KPTSSELQKM

[0420] 860 870 880 890 900

[0421] QEKQKLIKEP GSGVPVVLIT TLLVI PVWL LAIAI FIRWK KSRAFGADSE

[0422] 910 920 930 940 950

[0423] HKLETS SGRV LGRFRGKGSG GLNLGNFFAS RKGYSRKGFD RLSTEGSDQE 960 970

[0424] KEDDGSESEE EYSAPLPALA PSSS

[0425] SEQ ID No: 24 - Prepro-PAM Isoform 6 (amino acids 897-914 of SEQ ID No. 1 missing)

[0426] 10 20 30 40 50

[0427] MAGRVPSLLV LLVFPSSCLA FRSPLSVFKR FKETTRPFSN ECLGTTRPVV 60 70 80 90 100

[0428] PI DS SDFALD IRMPGVTPKQ SDTYFCMSMR I PVDEEAFVI DFKPRASMDT 110 120 130 140 150

[0429] VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA NILYAWARNA PPTRLPKGVG 160 170 180 190 200

[0430] FRVGGETGSK YFVLQVHYGD I SAFRDNNKD CSGVSLHLTR LPQPLIAGMY 210 220 230 240 250

[0431] LMMSVDTVI P AGEKVVNSDI SCHYKNYPMH VFAYRVHTHH LGKWSGYRV 260 270 280 2 90 300

[0432] RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF GDLLAARCVF TGEGRTEATH 310 320 330 340 350

[0433] IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT QNVAPDMFRT I PPEANI PI P 360 370 380 390 400

[0434] VKSDMVMMHE HHKETEYKDK I PLLQQPKRE EEEVLDQGDF YSLLSKLLGE 410 420 430 440 450

[0435] REDVVHVHKY NPTEKAESES DLVAEIANVV QKKDLGRSDA REGAEHERGN 460 470 480 4 90 500

[0436] AILVRDRIHK FHRLVSTLRP PESRVFSLQQ PPPGEGTWEP EHTGDFHMEE 510 520 530 540 550

[0437] ALDWPGVYLL PGQVSGVALD PKNNLVI FHR GDHVWDGNSF DSKFVYQQIG 560 570 580 590 600

[0438] LGPIEEDT IL VI DPNNAAVL QSSGKNLFYL PHGLS I DKDG NYWVTDVALH 610 620 630 640 650

[0439] QVFKLDPNNK EGPVLILGRS MQPGSDQNHF CQPTDVAVDP GTGAIYVSDG 660 670 680 690 700

[0440] YCNSRIVQFS PSGKFITQWG EESSGSSPLP GQFTVPHSLA LVPLLGQLCV 710 720 730 740 750

[0441] ADRENGRIQC FKTDTKEFVR EIKHSSFGRN VFAI SYI PGL LFAVNGKPHF 7 60 770 780 7 90 800

[0442] GDQEPVQGFV MNFSNGE I ID I FKPVRKHFD MPHDIVASED GTVYIGDAHT 810 820 830 840 850 NTVWKFTLTE KLEHRSVKKA GIEVQE IKEA EAWETKMEN KPTSSELQKM 860 870 880 890 900

[0443] QEKQKLIKEP GSGVPVVLIT TLLVI PVWL LAIAI FIRWK KSRAFGGKGS 910 920 930 940 950

[0444] GGLNLGNFFA SRKGYSRKGF DRLSTEGSDQ EKEDDGSESE EEYSAPLPAL

[0445] APSSS

[0446] SEQ ID No: 25 - PHM subunit of PAM

[0447] 10 20 30 40 50

[0448] FKETTRPFSN ECLGTTRPW PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR 60 70 80 90 100

[0449] I PVDEEAFVI DFKPRASMDT VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA 110 120 130 140 150

[0450] NILYAWARNA PPTRLPKGVG FRVGGETGSK YFVLQVHYGD I SAFRDNNKD 160 170 180 190 200

[0451] CSGVSLHLTR LPQPLIAGMY LMMSVDTVI P AGEKVVNSDI SCHYKNYPMH 210 220 230 240 250

[0452] VFAYRVHTHH LGKVVSGYRV RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF 260 270 280 2 90 300

[0453] GDLLAARCVF TGEGRTEATH IGGTS SDEMC NLYIMYYMEA KHAVSFMTCT 310 320 330 340 350

[0454] QNVAPDMFRT I PPEANI PI P VKSDMVMMHE HHKETEYKDK I PLLQQPKRE 360 370 380 390 400

[0455] EEEVLDQGDF YSLLSKLLGE REDWHVHKY NPTEKAESES DLVAEIANW 410 420 430 440 450

[0456] QKKDLGRSDA REGAEHERGN AILVRDRIHK FHRLVSTLRP PESRVFSLQQ

[0457] 460

[0458] PPPGEGTWEP EHTG

[0459] SEQ ID No: 26 - PAL subunit of PAM

[0460] 10 20 30 40 50

[0461] □FHMEEALDW PGVYLLPGQV SGVALDPKNN LVI FHRGDHV WDGNSFDSKF

[0462] 60 70 80 90 100

[0463] VYQQIGLGPI EEDTILVI DP NNAAVLQSSG KNLFYLPHGL SIDKDGNYWV

[0464] 110 120 130 140 150

[0465] TDVALHQVFK LDPNNKEGPV LILGRSMQPG SDQNHFCQPT DVAVDPGTGA

[0466] 160 170 180 190 200

[0467] IYVSDGYCNS RIVQFSPSGK FITQWGEESS GSSPLPGQFT VPHSLALVPL

[0468] 210 220 230 240 250

[0469] LGQLCVADRE NGRIQCFKTD TKEFVRE IKH SSFGRNVFAI SYI PGLLFAV

[0470] 260 270 280 290 300

[0471] NGKPHFGDQE PVQGFVMNFS NGE I IDIFKP VRKHFDMPHD IVASEDGTVY 310 320

[0472] IGDAHTNTVW KFTLTEKLEH RSV SEQ ID No: 27 - Sequence of recombinant human PAM 10 20 30 40 50

[0473] SPLSVFKRFK ETTRPFSNEC LGTTRPVVPI DSSDFALDIR MPGVTPKQSD

[0474] 60 70 80 90 100

[0475] TYFCMSMRIP VDEEAFVIDF KPRASMDTVH HMLLFGCNMP SSTGSYWFCD 110 120 130 140 150

[0476] EGTCTDKANI LYAWARNAPP TRLPKGVGFR VGGETGSKYF VLQVHYGDIS 160 170 180 190 200

[0477] AFRDNNKDCS GVSLHLTRLP QPLIAGMYLM MSVDTVIPAG EKWNSDISC 210 220 230 240 250

[0478] HYKNYPMHVF AYRVHTHHLG KWSGYRVRN GQWTLIGRQS PQLPQAFYPV 260 270 280 290 300

[0479] GHPVDVSFGD LLAARCVFTG EGRTEATHIG GTSSDEMCNL YIMYYMEAKH 310 320 330 340 350

[0480] AVSFMTCTQN VAPDMFRTIP PEANIPIPVK SDMVMMHEHH KETEYKDKIP

[0481] 360 370 380 390 400

[0482] LLQQPKREEE EVLDQGDFYS LLSKLLGERE DVVHVHKYNP TEKAESESDL 410 420 430 440 450

[0483] VAEIANWQK KDLGRSDARE GAEHERGNAI LVRDRIHKFH RLVSTLRPPE 460 470 480 490 500

[0484] SRVFSLQQPP PGEGTWEPEH TGDFHMEEAL DWPGVYLLPG QVSGVALDPK 510 520 530 540 550

[0485] NNLVIFHRGD HVWDGNSFDS KFVYQQIGLG PIEEDTILVI □PNNAAVLQS

[0486] 560 570 580 590 600

[0487] SGKNLFYLPH GLSIDKDGNY WVTDVALHQV FKLDPNNKEG PVLILGRSMQ 610 620 630 640 650

[0488] PGSDQNHFCQ PTDVAVDPGT GAIYVSDGYC NSRIVQFSPS GKFITQWGEE

[0489] 660 670 680 690 700

[0490] SSGSSPLPGQ FTVPHSLALV PLLGQLCVAD RENGRIQCFK TDTKEFVREI 710 720 730 740 750

[0491] KHSSFGRNVF AISYIPGLLF AVNGKPHFGD QEPVQGFVMN FSNGEI IDIF 760 770 780 790 800

[0492] KPVRKHFDMP HDIVASEDGT VYIGDAHTNT VWKFTLTEKL EHRSVKKAGI 810

[0493] EVQEIKEAEA VVGS

[0494] SEQ ID No: 28 - PHM fragment (aa 31-377 of PAM SEQ ID No. 1) 10 20 30 40 50

[0495] FKETTRPFSN ECLGTTRPW PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR

[0496] 60 70 80 90 100

[0497] IPVDEEAFVI DFKPRASMDT VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA 110 120 130 140 150

[0498] NILYAWARNA PPTRLPKGVG FRVGGETGSK YFVLQVHYGD ISAFRDNNKD 160 170 180 190 200

[0499] CSGVSLHLTR LPQPLIAGMY LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH 210 220 230 240 250

[0500] VFAYRVHTHH LGKVVSGYRV RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF 260 270 280 290 300 GDLLAARCVF TGEGRTEATH IGGTS SDEMC NLYIMYYMEA KHAVSFMTCT

[0501] 310 320 330 340

[0502] QNVAPDMFRT I PPEANI PI P VKSDMVMMHE HHKETEYKDK I PLLQQP

[0503] SEQ ID NO: 29 - PAM isoform 1 AS 31-973 ofPrepro-PAM Isoform 1 (amino acids 1-30 of SEQ ID

[0504] No. 19 missing)

[0505] 10 20 30 40 50

[0506] FKETTRPFSN ECLGTTRPW PI DSSDFALD IRMPGVTPKQ SDTYFCMSMR 60 70 80 90 100

[0507] I PVDEEAFVI DFKPRASMDT VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA

[0508] 110 120 130 140 150

[0509] NILYAWARNA PPTRLPKGVG FRVGGETGSK YFVLQVHYGD I SAFRDNNKD 160 170 180 190 200

[0510] CSGVSLHLTR LPQPLIAGMY LMMSVDTVI P AGEKVVNSDI SCHYKNYPMH 210 220 230 240 250

[0511] VFAYRVHTHH LGKVVSGYRV RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF 260 270 280 2 90 300

[0512] GDLLAARCVF TGEGRTEATH IGGTS SDEMC NLYIMYYMEA KHAVSFMTCT

[0513] 310 320 330 340 350

[0514] QNVAPDMFRT I PPEANI PI P VKSDMVMMHE HHKETEYKDK I PLLQQPKRE

[0515] 360 370 380 390 400

[0516] EEEVLDQGDF YSLLSKLLGE REDWHVHKY NPTEKAESES DLVAEIANW 410 420 430 440 450

[0517] QKKDLGRSDA REGAEHERGN AILVRDRIHK FHRLVSTLRP PESRVFSLQQ 460 470 480 4 90 500

[0518] PPPGEGTWEP EHTGDFHMEE ALDWPGVYLL PGQVSGVALD PKNNLVI FHR

[0519] 510 520 530 540 550

[0520] GDHVWDGNSF DSKFVYQQIG LGPIEEDTIL VIDPNNAAVL QS SGKNLFYL 560 570 580 590 600

[0521] PHGLS IDKDG NYWVTDVALH QVFKLDPNNK EGPVLILGRS MQPGSDQNHF 610 620 630 640 650

[0522] CQPTDVAVDP GTGAIYVSDG YCNSRIVQFS PSGKFITQWG EESSGS SPLP 660 670 680 690 700

[0523] GQFTVPHSLA LVPLLGQLCV ADRENGRIQC FKTDTKEFVR E IKHSSFGRN 710 720 730 740 750

[0524] VFAI SYI PGL LFAVNGKPHF GDQEPVQGFV MNFSNGEI I D I FKPVRKHFD

[0525] 7 60 770 780 7 90 800

[0526] MPHDIVASED GTVYIGDAHT NTVWKFTLTE KLEHRSVKKA GIEVQE IKEA

[0527] 810 820 830 840 850

[0528] EAWETKMEN KPTSSELQKM QEKQKLIKEP GSGVPWLI T TLLVI PWVL

[0529] 860 870 880 890 900

[0530] LAIAI FIRWK KSRAFGDSEH KLETS SGRVL GRFRGKGSGG LNLGNFFASR 910 920 930 940 943

[0531] KGYSRKGFDR LSTEGSDQEK EDDGSESEEE YSAPLPALAP S SS

[0532] SEQ ID NO: 30 - PAM isoform 2 AS 31-868 of prepro-PAM isoform 2 (amino acids 1-30 of SEQ ID

[0533] No. 20 missing) 10 20 30 40 50

[0534] FKETTRPFSN ECLGTTRPW PI DSSDFALD IRMPGVTPKQ SDTYFCMSMR 60 70 80 90 100

[0535] I PVDEEAFVI DFKPRASMDT VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA

[0536] 110 120 130 140 150

[0537] NILYAWARNA PPTRLPKGVG FRVGGETGSK YFVLQVHYGD I SAFRDNNKD 160 170 180 190 200

[0538] CSGVSLHLTR LPQPLIAGMY LMMSVDTVI P AGEKWNSDI SCHYKNYPMH 210 220 230 240 250

[0539] VFAYRVHTHH LGKVVSGYRV RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF 260 270 280 2 90 300

[0540] GDLLAARCVF TGEGRTEATH IGGTS SDEMC NLYIMYYMEA KHAVSFMTCT

[0541] 310 320 330 340 350

[0542] QNVAPDMFRT I PPEANI PI P VKSDMVMMHE HHKETEYKDK I PLLQQPKRE

[0543] 360 370 380 390 400

[0544] EEEVLDQDFH MEEALDWPGV YLLPGQVSGV ALDPKNNLVI FHRGDHVWDG 410 420 430 440 450

[0545] NSFDSKFVYQ QIGLGPIEED T ILVIDPNNA AVLQSSGKNL FYLPHGLS I D

[0546] 460 470 480 4 90 500

[0547] KDGNYWVTDV ALHQVFKLDP NNKEGPVLIL GRSMQPGSDQ NHFCQPTDVA 510 520 530 540 550

[0548] VDPGTGAIYV SDGYCNSRIV QFS PSGKFIT QWGEESSGS S PLPGQFTVPH 560 570 580 590 600

[0549] SLALVPLLGQ LCVADRENGR IQCFKTDTKE FVRE IKHSSF GRNVFAI SYI 610 620 630 640 650

[0550] PGLLFAVNGK PHFGDQEPVQ GFVMNFSNGE I IDIFKPVRK HFDMPHDIVA

[0551] 660 670 680 690 700

[0552] SEDGTVYIGD AHTNTVWKFT LTEKLEHRSV KKAGIEVQE I KEAEAVVETK

[0553] 710 720 730 740 750

[0554] MENKPTSSEL QKMQEKQKLI KEPGSGVPVV LITTLLVI PV WLLAIAIFI 7 60 770 780 790 800

[0555] RWKKSRAFGD SEHKLETS SG RVLGRFRGKG SGGLNLGNFF ASRKGYSRKG 810 820 830 836

[0556] FDRLSTEGSD QEKEDDGSES EEEYSAPLPA LAPSSS

[0557] SEQ ID No: 31 - PAM isoform 3 AS 31-905 of prepro-PAM isoform 3 (amino acids 1-30 of SEQ ID No. 21 missing)

[0558] 10 20 30 40 50

[0559] FKETTRPFSN ECLGTTRPW PI DSSDFALD IRMPGVTPKQ SDTYFCMSMR

[0560] 60 70 80 90 100

[0561] I PVDEEAFVI DFKPRASMDT VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA

[0562] 110 120 130 140 150

[0563] NILYAWARNA PPTRLPKGVG FRVGGETGSK YFVLQVHYGD I SAFRDNNKD

[0564] 160 170 180 190 200

[0565] CSGVSLHLTR LPQPLIAGMY LMMSVDTVI P AGEKWNSDI SCHYKNYPMH

[0566] 210 220 230 240 250

[0567] VFAYRVHTHH LGKVVSGYRV RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF 260 270 280 2 90 300

[0568] GDLLAARCVF TGEGRTEATH IGGTS SDEMC NLYIMYYMEA KHAVSFMTCT

[0569] 310 320 330 340 350

[0570] QNVAPDMFRT I PPEANI PI P VKSDMVMMHE HHKETEYKDK I PLLQQPKRE

[0571] 360 370 380 390 400

[0572] EEEVLDQGDF YSLLSKLLGE REDWHVHKY NPTEKAESES DLVAEIANW 410 420 430 440 450

[0573] QKKDLGRSDA REGAEHERGN AILVRDRIHK FHRLVSTLRP PESRVFSLQQ 460 470 480 4 90 500

[0574] PPPGEGTWEP EHTGDFHMEE ALDWPGVYLL PGQVSGVALD PKNNLVIFHR 510 520 530 540 550

[0575] GDHVWDGNSF DSKFVYQQIG LGPIEEDTIL VI DPNNAAVL QSSGKNLFYL

[0576] 560 570 580 590 600

[0577] PHGLS IDKDG NYWVTDVALH QVFKLDPNNK EGPVLILGRS MQPGSDQNHF

[0578] 610 620 630 640 650

[0579] CQPTDVAVDP GTGAIYVSDG YCNSRIVQFS PSGKFI TQWG EESSGSSPLP

[0580] 660 670 680 690 700

[0581] GQFTVPHSLA LVPLLGQLCV ADRENGRIQC FKTDTKEFVR E IKHSSFGRN 710 720 730 740 750

[0582] VFAI SYI PGL LFAVNGKPHF GDQEPVQGFV MNFSNGEI I D IFKPVRKHFD

[0583] 7 60 770 780 7 90 800

[0584] MPHDIVASED GTVYIGDAHT NTVWKFTLTE KLEHRSVKKA GIEVQEIKDS

[0585] 810 820 830 840 850

[0586] EHKLETSSGR VLGRFRGKGS GGLNLGNFFA SRKGYSRKGF DRLSTEGSDQ 860 870 885

[0587] EKEDDGSESE EEYSAPLPAL APSSS

[0588] SEQ ID No: 32 - PAM isoform 4 AS 31-887 of prepro-PAM isoform 4 (amino acids 1-30 of SEQ ID No. 22 missing) 10 20 30 40 50

[0589] FKETTRPFSN ECLGTTRPVV PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR

[0590] 60 70 80 90 100

[0591] I PVDEEAFVI DFKPRASMDT VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA

[0592] 110 120 130 140 150

[0593] NILYAWARNA PPTRLPKGVG FRVGGETGSK YFVLQVHYGD I SAFRDNNKD

[0594] 160 170 180 190 200

[0595] CSGVSLHLTR LPQPLIAGMY LMMSVDTVI P AGEKWNSDI SCHYKNYPMH

[0596] 210 220 230 240 250

[0597] VFAYRVHTHH LGKVVSGYRV RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF

[0598] 260 270 280 2 90 300

[0599] GDLLAARCVF TGEGRTEATH IGGTS SDEMC NLYIMYYMEA KHAVSFMTCT

[0600] 310 320 330 340 350

[0601] QNVAPDMFRT I PPEANI PI P VKSDMVMMHE HHKETEYKDK I PLLQQPKRE

[0602] 360 370 380 390 400

[0603] EEEVLDQGDF YSLLSKLLGE REDWHVHKY NPTEKAESES DLVAEIANW

[0604] 410 420 430 440 450

[0605] QKKDLGRSDA REGAEHERGN AILVRDRIHK FHRLVSTLRP PESRVFSLQQ 460 470 480 4 90 500

[0606] PPPGEGTWEP EHTGDFHMEE ALDWPGVYLL PGQVSGVALD PKNNLVIFHR 510 520 530 540 550

[0607] GDHVWDGNSF DSKFVYQQIG LGPIEEDTIL VIDPNNAAVL QS SGKNLFYL 560 570 580 590 600

[0608] PHGLS IDKDG NYWVTDVALH QVFKLDPNNK EGPVLILGRS MQPGSDQNHF

[0609] 610 620 630 640 650

[0610] CQPTDVAVDP GTGAIYVSDG YCNSRIVQFS PSGKFITQWG EESSGS SPLP

[0611] 660 670 680 690 700

[0612] GQFTVPHSLA LVPLLGQLCV ADRENGRIQC FKTDTKEFVR E IKHSSFGRN 710 720 730 740 750

[0613] VFAI SYI PGL LFAVNGKPHF GDQEPVQGFV MNFSNGEI I D I FKPVRKHFD 7 60 770 780 7 90 800

[0614] MPHDIVASED GTVYIGDAHT NTVWKFTLTE KLEHRSVKKA GIEVQEIKGK 810 820 830 840 850

[0615] GSGGLNLGNF FASRKGYSRK GFDRLSTEGS DQEKEDDGSE SEEEYSAPLP 857

[0616] ALAPSS S

[0617] SEQ ID No: 33 - PAM Isoform 5 AS 31-973 of prepro-PAM Isoform 5 (SEQ ID No. 23 with amino acids 1-30 missing)

[0618] 10 20 30 40 50

[0619] FKETTRPFSN ECLGTTRPW PI DSSDFALD IRMPGVTPKQ SDTYFCMSMR

[0620] 60 70 80 90 100

[0621] I PVDEEAFVI DFKPRASMDT VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA

[0622] 110 120 130 140 150

[0623] NILYAWARNA PPTRLPKGVG FRVGGETGSK YFVLQVHYGD I SAFRDNNKD

[0624] 160 170 180 190 200

[0625] CSGVSLHLTR LPQPLIAGMY LMMSVDTVI P AGEKVVNSDI SCHYKNYPMH

[0626] 210 220 230 240 250

[0627] VFAYRVHTHH LGKVVSGYRV RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF

[0628] 260 270 280 2 90 300

[0629] GDLLAARCVF TGEGRTEATH IGGTS SDEMC NLYIMYYMEA KHAVSFMTCT

[0630] 310 320 330 340 350

[0631] QNVAPDMFRT I PPEANI PI P VKSDMVMMHE HHKETEYKDK I PLLQQPKRE

[0632] 360 370 380 390 400

[0633] EEEVLDQGDF YSLLSKLLGE REDWHVHKY NPTEKAESES DLVAEIANW

[0634] 410 420 430 440 450

[0635] QKKDLGRSDA REGAEHERGN AILVRDRIHK FHRLVSTLRP PESRVFSLQQ

[0636] 460 470 480 4 90 500

[0637] PPPGEGTWEP EHTGDFHMEE ALDWPGVYLL PGQVSGVALD PKNNLVI FHR

[0638] 510 520 530 540 550

[0639] GDHVWDGNSF DSKFVYQQIG LGPIEEDTIL VIDPNNAAVL QS SGKNLFYL

[0640] 560 570 580 590 600

[0641] PHGLS IDKDG NYWVTDVALH QVFKLDPNNK EGPVLILGRS MQPGSDQNHF

[0642] 610 620 630 640 650

[0643] CQPTDVAVDP GTGAIYVSDG YCNSRIVQFS PSGKFITQWG EESSGS SPLP 660 670 680 690 700

[0644] GQFTVPHSLA LVPLLGQLCV ADRENGRIQC FKTDTKEFVR E IKHSSFGRN 710 720 730 740 750

[0645] VFAI SYI PGL LFAVNGKPHF GDQEPVQGFV MNFSNGEI I D I FKPVRKHFD 7 60 770 780 7 90 800

[0646] MPHDIVASED GTVYIGDAHT NTVWKFTLTE KLEHRSVKKA GIEVQE IKEA

[0647] 810 820 830 840 850

[0648] EAWETKMEN KPTSSELQKM QEKQKLIKEP GSGVPWLI T TLLVI PWVL 860 870 880 890 900

[0649] LAIAI FIRWK KSRAFGADSE HKLETSSGRV LGRFRGKGSG GLNLGNFFAS

[0650] 910 920 930 940 944

[0651] RKGYSRKGFD RLSTEGSDQE KEDDGSESEE EYSAPLPALA PSSS

[0652] SEQ ID No: 34 - PAM Isoform 6 AS 31-955 of prepro-PAM Isoform 6 (amino acids 1-30 of SEQ ID

[0653] No. 24 missing)

[0654] 10 20 30 40 50

[0655] FKETTRPFSN ECLGTTRPW PI DSSDFALD IRMPGVTPKQ SDTYFCMSMR 60 70 80 90 100

[0656] I PVDEEAFVI DFKPRASMDT VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA 110 120 130 140 150

[0657] NILYAWARNA PPTRLPKGVG FRVGGETGSK YFVLQVHYGD I SAFRDNNKD 160 170 180 190 200

[0658] CSGVSLHLTR LPQPLIAGMY LMMSVDTVI P AGEKVVNSDI SCHYKNYPMH 210 220 230 240 250

[0659] VFAYRVHTHH LGKVVSGYRV RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF 260 270 280 2 90 300

[0660] GDLLAARCVF TGEGRTEATH IGGTS SDEMC NLYIMYYMEA KHAVSFMTCT 310 320 330 340 350

[0661] QNVAPDMFRT I PPEANI PI P VKSDMVMMHE HHKETEYKDK I PLLQQPKRE 360 370 380 390 400

[0662] EEEVLDQGDF YSLLSKLLGE REDWHVHKY NPTEKAESES DLVAEIANW 410 420 430 440 450

[0663] QKKDLGRSDA REGAEHERGN AILVRDRIHK FHRLVSTLRP PESRVFSLQQ 460 470 480 4 90 500

[0664] PPPGEGTWEP EHTGDFHMEE ALDWPGVYLL PGQVSGVALD PKNNLVI FHR 510 520 530 540 550

[0665] GDHVWDGNSF DSKFVYQQIG LGPIEEDTIL VIDPNNAAVL QS SGKNLFYL 560 570 580 590 600

[0666] PHGLS IDKDG NYWVTDVALH QVFKLDPNNK EGPVLILGRS MQPGSDQNHF 610 620 630 640 650

[0667] CQPTDVAVDP GTGAIYVSDG YCNSRIVQFS PSGKFITQWG EESSGS SPLP 660 670 680 690 700

[0668] GQFTVPHSLA LVPLLGQLCV ADRENGRIQC FKTDTKEFVR E IKHSSFGRN 710 720 730 740 750

[0669] VFAI SYI PGL LFAVNGKPHF GDQEPVQGFV MNFSNGEI I D I FKPVRKHFD 7 60 770 780 7 90 800

[0670] MPHDIVASED GTVYIGDAHT NTVWKFTLTE KLEHRSVKKA GIEVQE IKEA 810 820 830 840 850

[0671] EAWETKMEN KPTSSELQKM QEKQKLIKEP GSGVPWLI T TLLVI PWVL 860 870 880 890 900

[0672] LAIAI FIRWK KSRAFGGKGS GGLNLGNFFA SRKGYSRKGF DRLSTEGSDQ 910 920 925

[0673] EKEDDGSESE EEYSAPLPAL APSSS

[0674] Literature

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[0676] 2. Tsuruda, T., Kato, J., Kuwasako, K. & Kitamura, K. Adrenomedullin: Continuing to explore cardioprotection. Peptides 111, 47-54 (2019).

[0677] 3. Kato, J. & Kitamura, K. Bench-to-bedside pharmacology of adrenomedullin. Eur. J. Pharmacol. 764, 140 148 (2015).

[0678] 4. Lewis, L. K. el al. Degradation of human adrenomedullin (1-52) by plasma membrane enzymes and identification of metabolites. Peptides 18, 733 739 (1997).

[0679] 5. Martinez, A. et al. Matrix metalloproteinase-2 cleavage of adrenomedullin produces a vasoconstrictor out of a vasodilator. Biochem. J. 383, 413 418 (2004).

[0680] 6. Nishimoto, Y., Nagata, S., Akashi, E., Yamasaki, M. & Kitamura, K. Thrombin rapidly digests adrenomedullin: Synthesis of adrenomedullin analogs resistant to thrombin. Biochem. Biophys. Res. Commun. 529, 778 783 (2020).

[0681] 7. Gibbons, C., Dackor, R., Dunworth, W., Fritz-Six, K. & Caron, K. M. Receptor activitymodifying proteins: RAMPing up adrenomedullin signaling. Mol. Endocrinol. 21, 783 796 (2007).

[0682] 8. Niu, P. et al. Accelerated cardiac hypertrophy and renal damage induced by angiotensin II in adrenomedullin knockout mice. Hypertens. Res. 26, 731 736 (2003).

[0683] 9. Nishikimi, T. et al. Chronic Administration of Adrenomedullin Attenuates Transition from Left Ventricular Hypertrophy to Heart Failure in Rats. Hypertension 42, 1034 1041 (2003).

[0684] 10. Okumura, H. et al. Adrenomedullin infusion attenuates myocardial ischemia / reperfusion injury through the phosphatidylinositol 3 -kinase / Akt-dependent pathway. Circulation 109, 242-248 (2004).

[0685] 11. Nakamura, R. et al. Adrenomedullin administration immediately after myocardial infarction ameliorates progression of heart failure in rats. Circulation 110, 426 431 (2004).

[0686] 12. Niu, P. et al. Protective Effects of Endogenous Adrenomedullin on Cardiac Hypertrophy, Fibrosis, and Renal Damage. Circulation 109, 1789 1794 (2004).

[0687] 13. Looi, Y. H., Kane, K. A., McPhaden, A. R. & Wainwright, C. L. Adrenomedullin acts via nitric oxide and peroxynitrite to protect against myocardial ischaemia-induced arrhythmias in anaesthetized rats. Br. J. Pharmacol. 148, 599 609 (2006).

[0688] 14. Yoshizawa, T. et al. Effects of adrenomedullin on doxorubicin-induced cardiac damage in mice. Biol. Pharm. Bull. 39, 737 746 (2016).

[0689] 15. Li, L. L. et al. Mesenchymal stem cells overexpressing adrenomedullin improve heart function through antifibrotic action in rats experiencing heart failure. Mol. Med. Rep. 17, 1437 1444 (2018).

[0690] 16. Nishikimi, T. et al. Effects of long-term intravenous administration of adrenomedullin (AM) plus hANP therapy in acute decompensated heart failure - A pilot study. Circ. J. 73, 892-898 (2009).

[0691] 17. Nagaya, N. et al. Haemodynamic and hormonal effects of adrenomedullin in patients with pulmonary hypertension. Heart 84, 653 658 (2000).

[0692] 18. Ashizuka, S., Kita, T., Inatsu, H. & Kitamura, K. Adrenomedullin: A Novel Therapeutic for the Treatment of Inflammatory Bowel Disease. Biomedicines 9, (2021).

[0693] 19. Garcia, M. A. et al. Adrenomedullin: a new and promising target for drug discovery. Expert Opin. Ther. Targets 10, 303 317 (2006).

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Increased plasma levels of adrenomedullin in patients with systemic inflammatory response syndrome. Am. J. Respir. Crit. Care Med. 160, 132 136 (1999). Ashizuka, S. et al. Adrenomedullin: A Novel Therapy for Intractable Crohn’s Disease with a Loss of Response to Infliximab. Intern. Med. 58, 1573 1576 (2019). Maki, T., Ihara, M. & Fujita, Y. Angiogenic and Vasoprotective Effects of Adrenomedullin on Prevention of Cognitive Decline After Chronic Cerebral Hypoperfusion in Mice. 1122-1128 (2011). doi:10.1161 / STROKEAHA.l 10.603399 Abbott, N. J., Patabendige, A. A. K., Dolman, D. E. M., Yusof, S. R. & Begley, D. J. Structure and function of the blood-brain barrier. Neurobiol. Dis. 37, 13 25 (2010). Hawkins, B. T. & Davis, T. P. The blood-brain barrier / neurovascular unit in health and disease. Pharmacol. Rev. 57, 173 185 (2005). Begley, D. J. & Brightman, M. W. Structural and functional aspects of the blood-brain barrier. Pept. Transp. Deliv. into Cent. Nerv. Syst. 39 78 (2003). 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L. Experimental colitis increases blood-brain barrier permeability in rabbits. Am. J. Physiol. Liver Physiol. 276, G1174 G1180 (1999). Hernandez, L. et al. Blood brain barrier and gut barrier dysfunction in chronic kidney disease with a focus on circulating biomarkers and tight junction proteins. Sci. Rep. 12, 4414 (2022). Momenabadi, S. et al. Oxytocin reduces brain injury and maintains blood brain barrier integrity after ischemic stroke in mice. Neuromolecular Med. 22, 557 571 (2020). Yang, J., Yang, C., Yang, Y., Jia, N. & Sun, Q. Protection of vasoactive intestinal peptide on the blood-brain barrier dysfunction induced by focal cerebral ischemia in rats. J. Stroke Cerebrovasc. Dis. 31, 106160 (2022). Korkmaz, O. T. et al. Vasoactive intestinal peptide decreases P-amyloid accumulation and prevents brain atrophy in the 5xFAD mouse model of Alzheimer’s disease. J. Mol. Neurosci. 68, 389 396 (2019). Rat, D. et al. Neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) slows down Alzheimer’s disease-like pathology in amyloid precursor protein-transgenic mice. FASEB J. 25, 3208 (2011). Wand, G. S., Ney, R. L., Mains, R. E. & Eipper, B. A. Characterization of peptide alphaamidation activity in human cerebrospinal fluid and central nervous system tissue. Neuroendocrinology 41, 482-489 (1985). Gether, U., Aakerlund, L. & Schwartz, T. W. Comparison of peptidyl-glycine a-amidation activity in medullary thyroid carcinoma cells, pheochromocytomas, and serum. Mol. Cell. Endocrinol. 79, 53 63 (1991). Wand, G. S., Nev, R. L., Mainsh, R. E. & Eipperh, B. A. Characterization of Peptide AlphaAmidation Activity in Human Cerebrospinal Fluid and Central Nervous System Tissue. Neuroendocronology 41, 482-489 (1985). Wand, G. S. et al. Alzheimer’s disease: Low levels of peptide a-amidation activity in brain and CSF. Neurology 37, 1057 1061 (1987). Tsukamoto, T. et al. Increased Peptidylglycine alpha-Amidating Monooxygenase Activity in Cerebrospinal Fluid of Patients with Multiple Sclerosis. Intern. Med. 34, 229 232 (1995). Ashizuka, S., Inatsu, H., Kita, T. & Kitamura, K. Adrenomedullin therapy in patients with refractory ulcerative colitis: a case series. Dig. Dis. Sci. 61, 872 880 (2016). Kataoka, Y. et al. The first clinical pilot study of intravenous adrenomedullin administration in patients with acute myocardial infarction. J. Cardiovasc. Pharmacol. 56, 413 419 (2010). Troughton, R. W. et al. Adrenomedullin Infusion in Essential Hypertension. 588 593 (2000). doi:10.1161 / 01.HYP.36.4.588 Nagaya, N. & Kangawa, K. Adrenomedullin in the treatment of pulmonary hypertension. Peptides 25, 2013 2018 (2004). Nagata, S., Yamasaki, M. & Kitamura, K. Peptides Polyethylene glycol-conjugated human adrenomedullin as a possible treatment for vascular dementia. Peptides 121, 170133 (2019). Dschietzig, T. et al. The adrenomedullin receptor acts as clearance receptor in pulmonary circulation. Biochem. Biophys. Res. Commun. 294, 315 318 (2002). Ertmer, C. et al. Exogenous adrenomedullin reduces the arterial lactate concentration and mean pulmonary arterial pressure in ovine endotoxemia. Crit. Care 11, 1 (2007). Burstein, B. et al. Association between mean arterial pressure during the first 24 hours and hospital mortality in patients with cardiogenic shock. Crit. Care 24, 513 (2020). Deniau, B., Takagi, K., Asakage, A. & Mebazaa, A. Adrecizumab: An investigational agent for the biomarker-guided treatment of sepsis. Expert Opin. Investig. Drugs 30, 95 102 (2021). Karakas, M. et al. Targeting Endothelial Dysfunction in Eight Extreme-Critically Ill Patients with COVID- 19 Using the Anti-Adrenomedullin Antibody Adrecizumab (HAM8101). Biomolecules 10, (2020). Yoshimoto, T., Saito, S. & Omae, K. 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[0695] Example / : ADM Measurements

[0696] Quantification of bio-ADM was conducted using the sphingotest bio-ADM assay as described elsewhere (Weber, J. et al., J. Appl. Lab. Med. An AACC Publ. 2, 222-233 (2017))'. 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 mMNaCl, 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.

[0697] 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 lh 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). 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 HAM1 101, pH 8.0, and reconstituted in H2O before use.

[0698] The molar concentration of the bio-ADM calibrator peptide preparation may be calculated from the mass concentration of the peptide preparation using the molecular weight of bio-ADM, wherein the mass concentration results from dissolving a specific mass of the bio-ADM preparation in a defined volume of a solvent. 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 a peptide preparation may vary from the calculated molecular weight in dependence on the used counter ion in the synthesis of the peptide and the number of basic amino acids and free amides within of the peptide sequence the counter ion binds to. A counter ion might be, but is not limited to, trifluoroacetic acid (TFA) with the molecular weight of 114.02 Da. The molecular weight (MW) of a synthetic peptide may be calculated as follows: MWpCptidc= (sum of all atoms in the peptide sequence) + x*MWCOuntcrion, wherein x is the number of free amides including basic amino acids within of peptides sequence. The molecular weight of bio-ADM may be additionally determined using mass spectrometry, in particular using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). A detailed procedure for the analysis of peptides containing disulfide bonds, as is the case in bio-ADM, using MALDI-TOF MS is described in (Huwiler KG, Mosher DF, Vestling MM. J Biomol Tech. 2003 Dec; 14(4): 289-97). Quantification of ADM-Gly was conducted as follows: 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 ADM-Gly (SEQ ID No.: 14, 17-21, 27) (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. 50 pL of samples / calibrators were pipetted into coated microtiter plates. Afterwards 200 pL of MACN labelled tracer antibody (AK835 / G4, directed towards the glycine extended C-terminus of ADM-Gly (SEQ ID No.: 14, 17-21, 27). AK835 / G4 had no cross-reactivity with bio-ADM) the microtiter plates were incubated for 18h at 4°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 ADM-Gly (SEQ ID No.: 14, 17-21, 27) (Peptides and Elephants, Hennigsdorf, Germany). The lowest calibrator did not contain ADM-Gly, but a concentration of 1 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. A typical standard curve is shown in Fig. 2A. A normal distribution of ADM-Gly from n=157 selfreported healthy individuals is shown in Fig. 2B. Using ADM-Gly values and bio-ADM values, the ratio of both peptides was calculated using formula (2).

[0699] Formula 2:

[0700] Example 2: Determination of PAM Activity (PAM-AMA)

[0701] 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 CuSCfi, 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.

[0702] 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 1. 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-t0min) 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.

[0703] A typical PAM calibration curve is shown in Fig. 3A._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 HC1 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 HC1, pH 7.5. Subsequently, 160 pL of reaction buffer (RB) (100 mM Tris-HCl, 6.25 pM CuSO4, 2.5 mM 1-ascorbate, 125 pg / mL bovine liver Catalase, 62.5 pM Amastatin, 125 pM 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 1. The concentration of generated bio-ADM was used to define the AMA of each calibrator-point (formula 3).

[0704] Formula 3:

[0705] DF: Dilution Factor of the sample

[0706] 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. 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.

[0707] 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. The distribution of AMA in Li-Heparin samples from n=120 self-reported healthy volunteers are shown in Fig. 3B. The median [IQR] of Li-Heparin AMA was 18.4 pg / (L*h) [13.5-21.9], The 10thand 90thpercentile were 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). In addition, matched serum samples from n=20 subjects were measured and revealed a highly significant correlation (r = 0.89; p < 0.0001); (Fig. 3C), although AMA values in serum were approximately 40% lower when compared to Li-Heparin.

[0708] Example 3: Determination of PAM concentration

[0709] Minimum 10 pL of samples / calibrators were pipetted into microtiter plates, pre-coated with a PAM specific antibody. After adding 200 pL of labelled, PAM specific tracer-antibody in buffer (300 mmol / L potassium phosphate, 100 mmol / L NaCl, 10 mmol / L Na-EDTA, 50 pmol / L amastatin, 100 pmol / L leupeptin, 0.1% bovine IgG, 0.02% mouse IgG, 0.5% BSA, pH 7.0) labelled with MACN, the microtiter plates were incubated for 3 h at room temperature (20 °C) under agitation at 600 rpm. Unbound tracer was removed by washing 5 times (each 350 pL per well) with washing solution (20 mmol / L 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).

[0710] Two-Step version: minimum 10 pL of samples / calibrators were pipetted into pre-coated microtiter plates. After adding 200 pL of buffer (as described in one-step version), the microtiter plates were incubated for 15-20 h at 2-8 °C under agitation at 600 rpm. Unbound sample was removed by washing 4 times (each 350 pL per well) with washing solution with subsequent addition of 200pl of tracer material and incubation of microtiter plates at room temperature (20 °C) for 2h. Unbound tracer was removed by washing 4 times (each 350 pL per well) with washing solution. Well-bound chemiluminescence was measured for 1 s per well by using the Centro LB 960 microtiter plate luminescence reader (Berthold Technologies).

[0711] A typical calibration curve for the LI A utilizing linear and conformational antibodies is shown in Fig. 4A, respectively. The distribution of PAM concentration (PAM-LIA) in serum samples from n=4106 individual (sub cohort of randomly selected individuals with no history of cardiovascular disease from the Swedish prospective population based study Malmo Preventive Project (MPP) is shown in Fig. 4B. The mean value of PAM-LIA was 77.8 ng / mL [SD=19.0]. The median plasma PAM concentration was

[0712] 78.6 ng / mL (inter quartile range [IQR] 66.4 92.5 ng / mL). The 10th and 90th percentile were 56.3 and

[0713] 106.6 ng / mL, respectively. The 2.5th, 97.5th and 99th percentile were 45, 123.5 and 135.5 ng / mL, respectively.

[0714] Example 4: Co-Infusion of ADM-Gly with Vitamin C

[0715] In the present invention either ADM-Gly (synthetic human 1-53 ADM-Gly (SEQ ID No.: 14) alone or ADM-Gly in combination with Vitamin C were infused in healthy rats (male Sprague Dawley rats). ADM-Gly was infused at a rate of 500 ng / (kg*min) over 150 min, resulting in a total dosage of 75 pg / kg. Vitamin C was infused at a rate of 1.5 mg / (kg*min) resulting in a total dosage of 225 mg / kg. When compounds were administered in combined manner, the dosage of each compound remained as described above. Animals were housed in temperature-controlled rooms (20-24°C) and maintained in a 12h light / 12h dark cycle. Animals were fed ad libitum before and during the study. The infusion was performed for 2.5 hours at a rate of 12.5 pl / min via a jugular vein catheter with n=3 animals per group. Blood was drawn before the infusions, as well as at 2.5h, 6h, and 12h after infusions were started. No signs of toxicity or any behavioural changes were observed during the observation period. To analyse the effect of infused preparation on generation of bio-ADM (SEQ ID No.: 15, 22-26, 28) from ADM- Gly (SEQ ID No.: 14), bio-ADM was determined in the collected plasma samples as described in Example 1 in comparison to baseline levels of bio-ADM in the animals.

[0716] When ADM-Gly was infused alone or in combination with Vitamin C, an increase of bio-ADM was observed due to amidation of infused ADM-Gly by endogenous PAM enzyme (Fig. 5). The generated bio-ADM levels reached 65 pg / mL (ADM-Gly alone) or 90 pg / mL (ADM-Gly + Vitamin C), but decreased after the infusion was stopped due to the short half-life of bio-ADM. The half-life time of the resulting bio-ADM elevation through amidation of ADM-Gly by endogenous circulating PAM is comparable to the half-life time of bio-ADM (and ADM-Gly in circulation as shown in Fig. 6), when bio-ADM is administered directly into the circulation (ca. 30 min). Surprisingly, the infusion of ADM- Gly (alone or together with vitamin C) showed no signs of toxicity or had any impact on behaviour of the animals or any other adverse effects.

[0717] These results present novel data, demonstrating that the administration of an inactive bio-ADM precursor, such as ADM-Gly, provides a non-toxic method for enriching bio-ADM. This enrichment can be further enhanced when co-administered with Vitamin C.

[0718] Example 5: Protective effect of ADM-Gly combination on Blood-Brain Barrier

[0719] Sepsis is the result of an acute and systemic immune response to a variety of noxious insults, in particular to bacterial infection. This response leads to the activation of a number of host mediator systems, including the cytokine, leukocyte, complement and haemostatic networks, each of which may contribute to the pathological sequelae of sepsis. In the sequence, the immune response can trigger vascular endothelial cells damage, interrupting tight junction’s proteins; consequently, the blood-brain barrier (BBB) breaks down, allowing and facilitating the entry of peripheral immune cells into the brain, which triggers or exacerbates the activation of glial cells and neuroinflammation.

[0720] 12-15 week old male C57B1 / 6N mice (Charles River, Germany) were used. All procedures were carried out according to the guidelines from the German Society for Animal Science (Gesellschaft fur Versuchstierkunde; GV-SOLAS).

[0721] Mice were anesthetized with isoflurane (induction of 3%, maintenance of 1.5%, and oxygen flow of 3 L / min). A 1-cm ventral midline abdominal incision was made and the cecum was ligated with 4-0 silk sutures distal to the ileocecal valve and punctured through with a 24-gauge needle. 2- to 3-mm droplet of fecal material was expelled. The incision was closed using 4-0 surgical sutures. Mice were fluid-resuscitated with 500 pl pre- warmed normal saline intraperitoneally immediately after the procedure. Sham animals underwent the same procedure except for CLP. For pre-operative treatment, 0.1 mg / kg buprenorphine sc + 100 mg / kg metamizole were administered parenterally. For post-operative analgesia, animals were injected s.c. with 0.1 mg / kg buprenorphine twice daily (morning and evening). Animals were folio wed- up for 24-hours, comprising n=12 mice per group (1 treatment group, 1 placebo group, 1 treatment-sham group, 1 placebo-sham group).

[0722] The reparative effect of ADM-Gly was tested in the treatment group. PBS was used in the Placebo groups. Placebo or the ADM-Gly were administered at day -1 before CLP via the intraperitoneal (i.p.) route of administration. The dosage of ADM-Gly was 50 pg / kg.

[0723] At the end of observation period (24 h) the animals were injected intravenously with the Evans Blue dye solution (4 mg / kg, dissolved in PBS at lmg / ml). The blue colour of extremities indicated that the dye has entered the bloodstream. In case of a functional BBB, the dye should not pass the BBB. In case of a disrupted BBB the dye will enter brain tissue. The mice were sacrificed 30 minutes later and to remove the dye from the blood vessels, a whole-body perfusion with PBS was performed through the left ventricle until the fluid from the right atrium was colourless. The brains were removed, photographed, weighted and homogenized in PBS. The homogenates were centrifuged for 20 min at 10,000 x g. The supernatants were measured for absorbance at 620 nm to quantify the presence of EB-dye using known EB-dye concentrations as standard.

[0724] Surprisingly, there was a strong and statistically significant effect of ADM-Gly treatment on the integrity of the blood brain barrier. In comparison to the placebo group (Fig. 7, black bars), the Evans-Blue extravasation in the group treated with ADM-Gly (Fig. 7, white bars, CLP treated) was significantly reduced and reached almost the same level to the SHAM treated animals (Fig. 7, white bars, SHAM). The resulting data clearly indicates, that a combined treatment with ADM-Gly (Fig. 7, white bar, CLP treated) had a preventive effect on the disruption of the BBB and therefore may be used to prevent or treat blood-brain barrier disruption in a series of pathologies, including, but not limited to, MCI, dementia, Alzheimer’s disease, stroke, sepsis and septic shock.

[0725] Example 6: Testing human or mouse adrenomedullin in human recombinant Adrenomedullin receptor cAMP functional assay (Adrenomedullin Bioassay) after modification of adrenomedullin by binding to antibodies.

[0726] Materials:

[0727] Cell line: CH0-K1

[0728] Receptor: Adrenomedullin (CRLR + RAMP3) Receptor Accession Number Cell line: CRLR: U17473; RAMP3: AJ001016

[0729] CH0-K1 cells expressing human recombinant adrenomedullin receptor (FAST-027C) grown prior to the test in media without antibiotic were detached by gentle flushing with PBS-EDTA (5 mM EDTA), recovered by centrifugation and resuspended in assay buffer (KRH: 5 mM KC1, 1.25 mM MgSO4, 124 mM NaCl, 25 mM HEPES, 13.3 mM Glucose, 1.25 mM KH2PO4, 1.45 mM CaC12, 0.5 g / 1 BSA).

[0730] Dose response curves were performed in parallel with the reference agonists (hADM or mADM).

[0731] Antagonist test (96well):

[0732] For antagonist testing, 6 pl of the reference agonist (human (5,63nM) or mouse (0,67nM) adrenomedullin) was mixed with 6 pl of the test samples at different antagonist dilutions; or with 6 pl buffer. After incubation for 60 min at room temperature, 12 pl of cells (2,500 cells / well) were added. The plates were incubated for 30 min at room temperature. After addition of the lysis buffer, percentage of DeltaF will be estimated, according to the manufacturer specification, with the HTRF kit from CisBio International (cat n°62AM2 PEB). hADM 22-52 was used as reference antagonist.

[0733] Antibodies testing cAMP-HTRF assay

[0734] The anti-h-ADM antibodies (NT-H, MR-H, CT-H) were tested for antagonist activity in human recombinant adrenomedullin receptor (FAST-027C) cAMP functional assay in the presence of 5.63nM Human ADM 1-52, at the following final antibody concentrations: lOOpg / ml, 20pg / ml, 4pg / ml, 0.8pg / ml, 0.16pg / ml.

[0735] The anti-m-ADM antibodies (NT-M, MR-M, CT-M) were tested for antagonist activity in human recombinant adrenomedullin receptor (FAST-027C) cAMP functional assay in the presence of 0.67nM Mouse ADM 1-50, at the following final antibody concentrations: lOOpg / ml, 20pg / ml, 4pg / ml, 0.8pg / ml, 0.16pg / tnl. Data were plotted relative inhibition vs. antagonist concentration (see figs. 3a to 31). The maximal inhibition by the individual antibody is given in table 1.

[0736] Table 1: Maximal inhibition by the respective antibody.

Claims

Claims1. ADM-Gly and / or a fragment thereof for use in the treatment of a disease in a subject, wherein said ADM-Gly and / or fragment thereof (SEQ ID No.: 4, 7-12) is in modified or unmodified form.

2. ADM-Gly and / or a fragment thereof for use in the treatment of a disease in a subject according to claim 1, wherein ADM-Gly and / or a fragment thereof is to be used in said subject that is in need of elevated level of bio-ADM and / or a fragment thereof (SEQ ID No.: 5, 13-18) in the circulation, wherein elevated level means by at least 10 pg / mL, preferably by at least 20 pg / mL, more preferably by at least 30 pg / mL, more preferably by at least 40 pg / mL, most preferably by at least 50 pg / mL.

3. ADM-Gly and / or a fragment thereof for use in the treatment of a disease in a subject according to claims 1 or 2, wherein said disease is characterized byA ratio between a) ADM-Gly and / or a fragment thereof (SEQ ID No.: 4, 7-12) and b) bio-ADM and / or a fragment thereof (SEQ ID No. 5, 13-18) above a threshold in a sample of bodily fluid of said subject and / or a bio-ADM and / or a fragment thereof (SEQ ID No. 5, 13-18) level below a certain threshold and / or an ADM-Gly and / or a fragment thereof (SEQ ID No.: 4, 7-12) level below a certain threshold and / or an ADM-Gly and / or a fragment thereof (SEQ ID No.: 4, 7-12) level above a certain threshold in combination with level of PAM and / or its isoforms (SEQ ID No. 29-34) and / or fragments thereof (SEQ ID No. 25-28) below a threshold and / or a level of PAM and / or its isoforms (SEQ ID No. 29-34) and / or fragments thereof (SEQ ID No. 25- 28) below a threshold in a sample of bodily fluid of said subject.

4. ADM-Gly and / or a fragment thereof for use in the treatment of a disease in a subject according to any of claims 1 to 3, wherein ADM-Gly and / or a fragment thereof is to be used in the treatment of the diseases selected from the group comprising pulmonary hypertension and hypertension, type-2 diabetes, inflammatory bowel disease and / or Crohn’s disease, sepsis and / or septic shock, cerebral infarction, mild cognitive impairment (MCI) and Alzheimer’s Disease (AD).

5. ADM-Gly and / or a fragment thereof for use in the treatment of a disease in a subject according to any of claims 1 to 4, wherein ADM-Gly and / or a fragment thereof (SEQ ID No.: 4, 7-12) is to be used in combination with Vitamin C.

6. ADM-Gly and / or a fragment thereof for use in the treatment of a disease in a subject according to any of claims 1 to 5, wherein the ratio between a) ADM-Gly and / or a fragment thereof (SEQ ID No.: 4, 7-12) and b) bio-ADM and / or a fragment thereof (SEQ ID No. 5, 13-18) in a bodily fluid is elevated over 1.5, preferably elevated over 1.6, more preferably elevated over 1.7, more preferably is elevated over 1.8, more preferable is elevated over 1.9, more preferably is higher than 2 and / or the concentration of PAM and / or its isoforms (SEQ ID No. 29-34) and / or fragments thereof (SEQ ID No. 25-28) in a bodily fluid is equal or below 100 ng / mL, preferably equal or below 90 ng / mL, more preferably equal or below 80 ng / mL, more preferably equal or below 60 ng / mL, more preferably equal or below 50 ng / mL and most preferably equal or below 40 ng / mL and / or the activity of PAM and / or its isoforms (SEQ ID No. 29-34) and / or fragments thereof (SEQ ID No. 25-28) in a bodily fluid is equal or below 24 Units / L, more preferably equal or below 20 Units / L, more preferably equal or below 16 Units / L, more preferably equal or below 14 Units / L, and most preferably equal or below 10 Units / L and / or bio-ADM and / or a fragment thereof (SEQ ID No.: 5, 13-18) is equal or below 20 pg / mL, preferably equal or below 15 pg / mL, preferably equal or below 10 pg / mL, preferably equal or below 5 pg / mL, and / orADM-Gly and / or a fragment thereof (SEQ ID No.: 4, 7-12) is equal or below 50 pg / mL, preferably equal or below 30 pg / mL, preferably equal or below 20 pg / mL, most preferred equal or below 15 pg / mL, and / orADM-Gly and / or a fragment thereof (SEQ ID No.: 4, 7-12) is equal or above 15 pg / mL, preferably equal or above 20 pg / mL, preferably equal or above 30 pg / mL, preferably is equal or above 50 pg / mL, together with the concentration of PAM and / or its isoforms (SEQ ID No. 29-34) and / or fragments thereof (SEQ ID No. 25-28) in a bodily fluid is equal or below 100 ng / mL, preferably equal or below 90 ng / mL, more preferably equal or below 80 ng / mL, more preferably equal or below 60 ng / mL, more preferably equal or below 50 ng / mL and most preferably equal or below 40 ng / mL, and / orADM-Gly and / or a fragment thereof (SEQ ID No.: 4, 7-12) is equal or above 15 pg / mL, preferably equal or above 20 pg / mL, preferably above 30 pg / mL, preferably is equal or above 50 pg / mL,together with the activity of PAM and / or its isoforms ( SEQ ID No. 29-34) and / or fragments thereof (SEQ ID No. 25-28) in a bodily fluid is equal or below 24 Units / L, more preferably equal or below 20 Units / L, more preferably equal or below 16 Units / L, more preferably equal or below 14 Units / L and most preferably equal or below 10 Units / L.

7. ADM-Gly and / or a fragment thereof for use in the treatment of a disease in a subject according to any of claims 1 to 6, wherein ADM-Gly is applied at a dosage of 0.1-500 pg / kg, preferably 1-400 pg / kg, more preferably 2-300 pg / mL, more preferably 2-300 pg / kg, more preferably 3-200 pg / kg, more preferably 5-100 pg / kg or wherein a fragment of ADM-Gly is applied at a corresponding molar dosage to said ADM-Gly .

8. ADM-Gly and / or a fragment thereof for use in the treatment of a disease in a subject according to any of claims 1 to 7, wherein ADM-Gly and / or a fragment thereof (SEQ ID No.: 4, 7-12) is to be used in combination 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.

9. ADM-Gly and / or a fragment thereof for use in the treatment of a disease in a subject according to any of claims 1 to 8, wherein said ADM-Gly and / or a fragment thereof (SEQ ID No.: 4, 7-12) is 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, more preferably via fusion with IgG Fc regions or Transferrin, most preferably via post-translational modifications attaching natural or synthetic polymers, wherein 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.

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