Compounds and methods for a long-lasting pam

Abstract

The present invention is directed to increase the level of peptidylglycine alpha-amidating monooxygenase (PAM) in human circulation for use as a long-acting medicament and the treatment of a subject, wherein said treatment comprises: reducing the potential or risk for a disease or disorder, and / or reducing the occurrence of a disease or disorder, and / or reducing the severity of a disease or disorder.

Description

[0001]P75545WO BOEHMERT & BOEHMERTCompounds and Methods for long-lasting PAM elevation The present invention is directed to compounds and methods for long-lasting peptidyl-glycinealpha-amidating monooxygenase (PAM) elevation in a subject compared to unmodified PAMas further described herein, wherein the enzymatic activity of modified PAM is maintained compared to unmodified PAM. The present invention is furthermore directed to corresponding medical applications and pharmaceutical compositions. The present invention further relates to compounds and methods in order to enrich PAM in asubject and / or an increase in the in vivo half-life of PAM, in particular in a subject, particularlyto achieve a long-lasting elevation of certain mature C-terminally amidated peptides, in particular bioactive peptide hormones, e.g. bio-active Adrenomedullin (bio-ADM), in vivo. Further embodiments of the invention are described herein below. Background of the invention Biologically active peptide hormones fulfill the function as signaling molecules. Most bioactivepeptide hormones are synthesized from larger, inactive precursor peptides (for an example seeFigure 1). During their biosynthesis, those peptides undergo several co- and posttranslationalmodifications, including cleavage of signal peptides, endoproteolytic cleavage of the precursorpro-peptides by specific endopeptidases mostly at pairs of basic residues, removal of basicresidues by carboxypeptidases, formations of disulfide bonds and N- and O-glycosylation(Eipper et al. 1993. Protein Science 2(4): 489–97). More than half of the known neural and endocrine peptides require an additional modification step to gain full biological activity involving the formation of a C-terminal alpha amide group (Guembe, et al.1999. J Histochem Cytochem 47(5): 623–36). This final step of peptide hormone biosynthesis involves the action of the bifunctional enzyme peptidylglycine alpha-amidating monooxygenase (PAM). PAM specifically recognizes C-terminal glycine residues in its substrates, cleaves glyoxylate from the peptide’s C-terminal glycine residue in a two-step enzymatic reaction leading to the formation of C-terminally alpha-amidated peptide hormones, wherein the resulting alpha-amide group originates from the cleaved c-terminal glycine (Prigge et al. 2004. Science 304(5672): 864–67). This amidation reaction takes place in the lumen of secretory granules prior to exocytosis of the amidated product (Martinez and Treston 1996. Molecular and CellularEndocrinol 123: 113–17). Alpha-amidated peptides are for example adrenomedullin (ADM),substance P, vasopressin, neuropeptide Y, Amylin, calcitonin, neurokinin A and others. However, previously it was demonstrated that PAM can also catalyze the formation of alpha- amides from glycinated substrates of non-peptide character, e.g. N-fatty acyl-glycines, which are converted by PAM to primary fatty acid amides (PFAMs) like oleamide. The identified and purified peptidyl-glycine amidating activities were shown to be dependent on copper and ascorbate (Emeson et al. 1984. Journal of Neuroscience: 2604–13; Kumar et al. 2016. J Mol Endocrinol 56(4):T63-76; Wand et al.1985. Neuroendocrinology 41: 482–89). 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 1-6). The PAM enzyme was found to be expressed at different levels in almost all mammalian cell types, with significant expression in airway epithelium, endothelial cells, ependymal cells in the brain, adult atrium, brain, kidney, pituitary, gastrointestinal tract and reproductive tissues (Chen et al. 2018. Diabetes Obes Metab 20 Suppl 2:64-76; Oldham et al. 1992. Biochem Biophys Res Commun 184(1): 323–29; Schafer et al.1992. J Neurosci 12(1): 222–34). The precursor protein (1-973 amino acids) of the largest known PAM Isoform 1 (SEQ ID No.1) encoded by the PAM cDNA is depicted in Figure 2. The N-terminal signal sequence or pre-peptide (amino acids 1-20) assures direction of the nascent PAM polypeptide into the secretorylumen of endoplasmic reticulum and is subsequently cleaved co-translationally. Afterwards the PAM-pro-peptide is processed by the same machinery used for the biosynthesis of integral membrane proteins and secreted proteins including cleavage of the pro-region (amino acids 21- 30), assuring proper folding, disulfide bond formation, phosphorylation and glycosylation(Bousquet-Moore et al.2010. J Neurosci Res 88(12):2535-45). The sequences of PAM isoforms1 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 as SEQ ID No.11 to 16 As depicted in Figure 2, the PAM cDNA further encodes two distinct enzymatic activities. The first enzymatic activity is named peptidyl-glycine alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3), is an enzyme, capable of catalyzing the conversion of a C-terminal glycine residue to an alpha hydroxy-glycine. The second activity is named peptidyl-a-hydroxy-glycine alpha-amidating lyase (PAL; EC 4.3.2.5) is an enzyme capable of catalyzing the conversion of an alpha hydroxy-glycine to an alpha-amide with subsequent glyoxylate release. The sequential action of these separate enzymatic activities results in the overall peptidyl-glycine alpha amidating activity. The first enzymatic activity (PHM) is located directly upstream of the pro- region (within amino acids 31-494 of isoform 1 (SEQ ID No.7)). The second catalytic activity (PAL) is located after exon 16 in isoform 1 within amino acids 495-817 (SEQ ID No.8). As depicted in Figure 2, both activities may be encoded together within of one polypeptide asa membrane-bound protein (isoforms 1, 2, 5, 6; corresponding to SEQ ID No. 1, 2, 5 and 6 ofprepro-PAM or corresponding to SEQ ID No.11, 12, 15 and 16 of PAM after cleavage of theprepro-sequence), as well within of one polypeptide as a soluble protein lacking thetransmembrane domain (TMD) (isoforms 3 and 4; corresponding to SEQ ID No. 3 and 4 ofprepro-PAM or corresponding to SEQ ID No. 13 and 14 of PAM after cleavage of the prepro-sequence). While isoforms 1, 2, 5 and 6 remain in the outer plasma membrane after fusion of secretory vesicles with the plasma membrane with subsequent endocytosis and recycling or degradation, soluble PAM isoforms lacking the TMD (isoforms 3 and 4) (amino acids 864-887) are co-secreted with the peptide-hormones (Wand et al. 1985 Metabolism 34(11): 1044–52). Furthermore, prohormone convertases may convert membrane bound PAM protein into soluble PAM protein by cleavage within the flexible region (exons 25 / 26) connecting PAL with the TMD during the secretory pathway (Bousquet-Moore et al.2010. J Neurosci Res 88(12):2535- 45). The PHM subunit may be cleaved from soluble or membrane bound PAM within the secretory pathway by prohormone convertases that address a double-basic cleavage-site in the exon 16 region. Furthermore, during endocytosis the full-length PAM protein may be alsoconverted into a soluble form due to the action of alpha- and gamma secretases (Bousquet-Moore et al. 2010. J Neurosci Res 88(12):2535-45). Membrane bound PAM from late endosome can be further secreted in form of exosomal vesicles. PHM and PAL activities, as well as the activity of the full-length PAM were determined in several human tissues and body fluids. However, the separated PHM and PAL activities in soluble forms will also lead to formation of c terminally alpha amidated products from c- terminally glycinated substrates when allowed to perform their separate reactions in the same compartment, body-fluid or in vitro experimental setup. How the transfer of the PHM hydroxylated product to the PAL takes place is not exactly understood to date. There is evidence that the hydroxylated product is released into solution and is not directly transferred from PHM to PAL (Yin et al. 2011. PLoS One 6(12):e28679). Additionally, the source of PAM incirculation remained unclear.The partial reaction of PHM is depicted in Figure 3. PHM is a copper dependent monooxygenase responsible for stereo-specific hydroxylation of the c-terminal glycine at the alpha carbon atom. During the hydroxylation reaction ascorbate is believed to be the naturally occurring reducing agent, while the oxygen in the newly formed hydroxyl group was shown to originate from molecular oxygen. The partial reaction of the PAL is depicted in Figure 3. The catalytic action of PAL involves proton abstraction form the PHM-formed hydroxy-glycine by a protein-backbone derived base and a nucleophilic attack of hydroxyl-group oxygen to the divalent metal leading to a cleavage of glyoxylate and formation of a c-terminal amide. PAM activity was analyzed in several human tissues and body fluids of healthy specimen or thosesuffering from several diseases. It has been shown that changes in PAM activity are linked todifferent pathologies. To summarize efforts that has been done in past:The presence of amidating activity in human cerebrospinal fluid (CSF) was shown by Wandand colleagues (Wand et al. 1985 Neuroendocrinol 41: 482–89). In patients suffering fromAlzheimer’s disease (AD) PAM activity in CSF were significantly decreased in comparison toactivities from normal specimen (Wand et al. 1987 Neurology 37: 1057–61). In addition, in WO2015 / 103594 the presence of PAM-Protein in CSF detected by mass spectrometry of AD-patients was proposed to be reduced compared to healthy controls. Moreover, bio-ADM, oneof the amidated products of PAM, was shown to be reduced in patients with prevalent andincident Alzheimer’s disease (WO2019 / 154900).Amidating activity in CSF of patients with low back pain was analyzed using 1-12 SubstanceP Gly (SP Gly) as substrate (Hyyppä et al.1990 Pain 43: 163–68). PAM activities of patientssuffering from multiple sclerosis (MS) were shown to be significantly decreased in serum(Tsukamoto et al. 1995. Internal Medicine 34(4): 229–32; WO2010 / 005387). An association between reduced plasma activity of PAM and type-2-diabetes was described in(WO2014 / 118634). Moreover, multiple PAM mutations resulting in reduced activity wereshown to be associated with an increased risk of type 2 diabetes, possibly by affecting insulin granule packaging and release from ß-cells (Thomsen et al.2018 Nat. Genet.50: 1122–1131;Chen et al. 2020: Diabetologia 63: 561–576; Sheng et al. 2022 Funct. Integr. Genomics 22:525–535) Patients suffering from multiple endocrine neoplasia type 1 (MEN-1) and pernicious anemia showed a decreased plasma PAM activity in comparison to healthy control subjects (Kapuscinski et al.1993. Clin Endocrinol 39(1): 51–58).In infectious diseases, such as sepsis and septic shock, the increase in PAM activity wasobserved (Example 1). Specifically, this elevation of PAM activity in prospective observationalmultinational Adrenomedullin and Outcome in Sepsis and Septic Shock-1 (AdrenOSS-1)cohort was predictive of 28-day mortality with a hazard ratio (HR) of 2.11 (p = 0.008) (Example1).Further analysis of the inactive ADM precursor, Adrenomedullin-Glycin (ADM-Gly), revealeda significantly higher ADM-Gly concentration in non-survivors compared to survivors (p <0.0001) (Example 2). Also here, high ADM-Gly concentrations strongly predict 28-daymortality with a HR of 3.28 (p<0.0001) (Example 2). These findings suggest that theadministration of PAM may be beneficial for patients in acute pathological conditions, particularly in cases where the conversion of inactive pre-hormones to active peptide hormonesis desired. The aforementioned examples suggest that the change in PAM activity is closelylinked with the progression of various diseases.Therefore, restoring PAM levels in pathological conditions as described above to the PAMlevels as observed in healthy state could potentially reverse the pathological state. Therefore, itis desirable to develop methods, which will achieve a long-lasting increase of PAM levels inhuman circulation, administering recombinant native PAM intravenously in vivo has beenshown to elevate PAM activity, leading to an increase in bio-ADM concentration in bloodcirculation. This effect is relatively short-lived due to the native enzyme's short average half-life in circulation of 47 minutes (Kaufmann et al. 2021, Scientific Reports (11): 1579).As mentioned above, PAM is the only known enzyme for the conversion of inactive hormones into their active form via C-terminal alpha amidation (Eipper et al., Annu Rev Neurosci 1992, 15: 57-85). More than half of known hormones require this process for their activation.Increasing the half-life of PAM in circulation could provide a way to increase the levels ofseveral amidated peptides, as opposed to increasing the level of each peptide individually usingdiverse stabilizing methods. This could have significant benefits both in terms of cost andefficiency of treatment, as well as potentially improving patient outcomes by increasing theavailability of active peptides.Brief description of the figuresFigure 1: Schematic representation of adrenomedullin processing pathway. Proadrenomedullin N-terminal 20 peptide (PAMP), midregional proadrenomedullin (MR- proADM), adrenomedullin with C-terminal Glycin (ADM-Gly) and C-terminal proadrenomedullin (CT-proADM) and are cleaved off the ADM precursor peptide, proadrenomedullin (proADM). The final step of ADM activation is catalyzed by peptidylglycine α-amidating monooxygenase (PAM). Figure 2: Schematic representation of PAM Isoform 1. Black bold arrows indicate cleavage- sites at double-basic amino-acids.Figure 3: Representative enzymatic reaction catalysed by PAM.Figure 4: PAM activity in sepsis and septic shock. PAM activity in AdrenOSS-1 subcohort. (A) Differences in PAM activity in Li-Heparin plasma measured upon admission to the ICU in patients with sepsis and septic shock, analyzed via one-way ANOVA (Dunn's correction) with control cohort comprising self-reported healthy individuals. Significance of differences is shown in each panel. (b) Twenty-eight-day Kaplan-Meier survival curves of low versus high PAM activity based on a cutoff value of 35.1 Units / L for PAM activity in patients with sepsis and septic shock.Figure 5: Determination of ADM-Gly concentration in sepsis and septic shock. ADM-Gly inAdrenOSS-1 subcohort. (A) Differences in concentration of ADM-Gly in Li-Heparin plasma measured upon admission to the ICU in patients with sepsis and septic shock, analyzed viaone-way ANOVA (Dunn's correction) with the control cohort comprising self-reportedhealthy individuals. Significance of differences is shown in each panel. (b) Kaplan-Meier survival curves over 28 days: Low vs High ADM-Gly concentration, with a cutoff value of 730 pg / ml in patients with sepsis and septic shock. Figure 6: Representative calibration curve of recombinant PAM (ADM maturation activity [AMA]). Figure 7: Frequency distribution (histogram) of AMA in self-reported healthy individuals (n=120). Figure 8: Characterization of PEGylated PAM by means of size exclusion chromatography and SDS-PAGE analysis. Figure 9: Comparative activity analysis of pegylated and unmodified PAM enzymes measured in three samples with PAM concentrations ranging from 2 µg / mL, 1 µg / mL, to 0.5 µg / mL. Figure 10: Single dose PAM injection. Comparison between PEG-PAM and unmodified PAM. Comparison of half-life times between PEG-PAM and unmodified PAM: A single-dose intravenous injection of 14.4 Units per kg of animal weight was administered, and blood samples were collected within 300 minutes after administration for analysis with respect to AMA. Figure 11: Enrichment of plasmatic AMA after s.c. bolus injection of PEG-PAM: A 984% increase in amidating activity in circulation, accessed by measuring AMA within 24 hours post-administration, was observed compared to the AMA baseline. Figure 12: Enrichment of plasmatic AMA after i.p. bolus injection of PEG-PAM: A 1900% increase in amidating activity in circulation, accessed by measuring AMA within 8 hours post-administration, was observed compared to the AMA baseline.Figure 13: Enrichment of plasmatic AMA after i.m. bolus injection of PEG-PAM: A 1680%increase in amidating activity in circulation, accessed by measuring AMA within 4 hours post-administration, was observed compared to the AMA baseline. Figure 14: Protective effect of PEG-PAM on blood-brain barrier integrity in a rodent sepsis model: Concentration of Evans Blue dye in brain homogenates was used as a measure of blood-brain barrier integrity. Black bars indicate animals treated with 1xPBS (placebo group). Grey bars indicate animals treated with PEG-PAM. Significance was tested via ordinary One- Way ANOVA using graph-pad prism version 7.0. n.s. not significant. Figure 15: Left: comparative activity analysis of pegylated or xtenylated and unmodifiedPAM enzymes. Right: comparative analysis of pegylated or xtenylated and unmodified PAMenzymes on an SDS-PAGE. Detailed Description of the Invention / Subject matter of the inventionSubject matter of the present invention are methods and compounds allowing for long-lastingnon-toxic elevation of PAM levels and / or levels of amidated peptides in blood circulation bymodified PAM. The term “PAM level” refers in particular to the PAM activity and / or PAM concentration, moreparticularly PAM activity, which in certain embodiments is determined by measuring PAMamidating activity or by a PAM immunoassay as described below, respectively. In the present invention, the amidating activity of the modified PAM is maintained; particularly at least 10% of the amidating activity are maintained, compared to unmodified PAM, more particularly, at least 20%, more particularly, at least 25%, more particularly, at least 50%, more particularly, at least 75%, more particularly, at least 80%, more particularly, at least 90%. The term “amidating activity”, “alpha-amidating activity”, “peptidyl-glycine alpha-amidatingactivity”, “AMA” or “PAM activity” refers to the sequential enzymatic activities of PHM andPAL, independent of the present splice variant or mixtures of splice variants or post- translationally modified PAM enzymes or soluble, separated PHM or PAL activities or soluble PHM and membrane bound PAL or combinations of all mentioned forms leading to the formation of alpha amidated products of peptide or non-peptide character, particularly ofpeptide character, from glycinated substrates of peptide or non-peptide character, particularlyof peptide character. In other words, the term “amidating activity”, “alpha-amidating activity”, “peptidyl-glycine alpha-amidating activity” or “PAM activity” may be described as the sequential action of enzymatic activities located within amino acids 21 to 817 in the propeptide encoded by the human PAM cDNA, independent of present splice-variants or mixtures thereof. The AMA of (modified) PAM can for example be determined in aqueous solution, e.g. abuffered solution, adding a buffered reaction solution comprising oxidases / peroxidasesenzymes such as catalase, protease inhibitors such as amastatin, leupeptin, reduction agent suchas ascorbate and ADM-Gly or another suitable alpha-Gly peptide and an antibody againstADM-Gly or said alpha-Gly peptide, and adding a second solution to stop the amidating reaction, e.g. a solution comprising a chelating agent such as EDTA, to fractions of the reaction solution at different time points to stop the reaction; the reaction product produced at different time points can then be quantified e.g. with a quantitative immunoassay. A particular such assay is described herein in Example 3. In certain embodiments the AMA can also be determined in vitro by measuring theconcentration of amidated (e.g. synthetic) peptides with C-terminal glycine over a definedperiod of time by detection methods selected from but not limited to radioimmunoassay (RIA),homogeneous enzyme-multiplied immunoassays (EMIT), chemiluminescence- andfluorescence-immunoassays, Enzyme-linked immunoassays (ELISA), Luminex-based bead arrays, protein microarray assays, and rapid test formats such as for instance immunochromatographic strip tests.PAM concentration can also be determined with a PAM immunoassay, such as an assayselected from the following: -One-Step version: samples / calibrators (particularly a minimum 10 μL of) are pipettedinto pre-coated vessels, particularly microtiter plates; after adding (particularly 200 μL) of labelled anti-PAM antibody in buffer (particularly 300 mmol / L potassium phosphate,100 mmol / L NaCl, 10 mmol / L Na-EDTA, 50 µmol / L amastatin, 100 µmol / L leupeptin, 0.1% bovine IgG, 0.02% mouse IgG, 0.5% BSA, pH 7.0), the microtiter plates are incubated (particularly for at least 3 h at room temperature (20 °C) under agitation at 600 rpm); unbound tracer is removed by washing (particularly 5 times, each 350 μL perwell) with washing solution (particularly 20 mmol / L PBS, 1 g / L Triton X-100, pH 7.4);well-bound chemiluminescence is measured, particularly for 1 s per well e.g. by usingthe Centro LB 960 microtiter plate luminescence reader (Berthold Technologies).- Two-Step version: of samples / calibrators (particularly a minimum 10 μL) are pipettedinto pre-coated vessels, particularly microtiter plates; after adding (particularly 200 μL of) buffer (as described in one-step version), the microtiter plates are incubated (particularly for 15-20 h at 2-8 °C under agitation at 600 rpm); unbound sample is removed by washing (particularly 4 times each 350 μL per well) with washing solution with subsequent addition of (particularly 200µl of) tracer material and incubation ofmicrotiter plates (particularly at room temperature (20 °C) for 2h); unbound tracer is removed by washing (particularly 4 times each 350 μL per well) with washing solution; well-bound chemiluminescence is measured (particularly for 1 s per well by using theCentro LB 960 microtiter plate luminescence reader (Berthold Technologies)).- The technology used is particularly a sandwich luminescence immunoassay, based onacridinium ester labelling;- Labelled compound (tracer) is particularly: Purified anti-PAM antibodies (0.2 g / L) arelabelled (particularly by incubation in 10% labelling buffer (particularly 500 mmol / L sodium phosphate, pH 8.0) with (particularly 1:5 mol / L ratio of) MACN-acridinium-NHS-ester (particularly 1 g / L, InVent GmbH), particularly for 20 min at 22 °C); afteradding buffer (particularly 5% 1 mol / L Tris-HCl, pH 8.0, for 10 min), the respective antibody is separated from free label (particularly via CentriPure P10 columns (emp Biotech GmbH)); the purified labelled antibody is diluted (particularly in 300 mmol / lpotassium phosphate, 100 mmol / l NaCl, 10 mmol / l Na-EDTA, 5 g / l Bovine Serum Albumin (pH 7.0)); the final concentration is particularly approximately 20 ng of labelled antibody per 150 μL.- Solid phase: vessels (particularly white polystyrene microtiter plates (Greiner Bio-OneInternational AG)) are coated (particularly 18 h at 20 °C) with the respective antibodyin buffer (particularly 2 μg / 0.2 mL per well 50 mmol / L Tris-HCl, 100 mmol / L NaCl,pH 7.8); After blocking (particularly with 30 g / L Karion, 5 g / L BSA (protease free), 6.5mmol / L monopotassium phosphate, 3.5 mmol / L sodium dihydrogen phosphate (pH 6.5)), the plates are vacuum-dried.- Calibration: The assay is calibrated, using dilutions of commercially purchasedrecombinant PAM (particularly according to Example 4, Variant B). The typical concentration range is within of 1 – 1,000 ng / mL. As used herein, PAM is in particular human PAM and more particularly includes all isoformsand single subunits thereof, in particular those generated by alternative splicing, which are inparticular the proteins comprising the polypeptide chains of SEQ ID Nos. 1 to 10.It is apparent to the skilled person that SEQ ID No.: 1 and SEQ ID No.: 2-6 as shown in thedescription of the respective sequences herein below are the prepro forms of PAM (containingthe pre- and the propeptide) and SEQ ID No. 10 relates to a pro-form fragment of PAM. Theskilled person is readily aware which parts of the sequence of the prepro or pro forms of PAMpertain to signal- or pro-peptides, and which parts pertain to mature PAM. Notwithstanding,the respective mature forms of PAM, which are preferred forms of PAM under the present invention, are shown for sake of completeness in SEQ ID No.: 11-16. For example, according to the present invention, PAM may mean PAM (SEQ ID No.: 11)and / or its fragments and / or isoforms (SEQ ID No.: 12 – 16, SEQ ID No.: 18), and / or catalyticsubunits like PHM (SEQ ID No.: 7, 9) and PAL (SEQ ID No.: 8), and / or fragments thereof in unmodified or modified form. This applies likewise, mutatis mutandis, to the further embodiments of the invention.SEQ ID No. 10 is a wildtype pro-fragment of PAM with a C-terminal amino acid extension oftwo amino acids G (Glycine) and S (serine). SEQ ID No. 18 is a wildtype fragment of PAMwith a C-terminal amino acid extension of two amino acids G (Glycine) and S (serine). In embodiments of the present invention, PAM is a recombinant PAM protein according to SEQ ID No.17.The term “unmodified PAM” refers to all isoforms and single subunits of PAM, in particularthose based on the SEQ ID Nos. 1 to 10 and which have not undergone any deliberate orartificial modifications, including chemical conjugation, alteration of the amino acid sequence,including amino acid exchange, deletion or insertion, fusion with other proteins, or other suchmethods. While there can be certain variations depending on its source organism, conditions ofexpression and / or purification, as well as certain naturally occurring mutants and variantsknown in the field, in particular unmodified PAM has an amino acid sequence of naturallyoccurring PAM and more particularly at least essentially retains its natural three-dimensional structure, function, and properties. Unmodified PAM may also refer to recombinant PAM (SEQ ID No.: 17). The term “modified PAM” refers to all isoforms and single subunits of PAM, in particular thosebased on the SEQ ID Nos. 1 to 10, which are modified in accordance with the present invention,in particular as detailed herein, more particularly by amino acid manipulation, by fusion to otherproteins, such as albumin, e.g. serum albumin or recombinant serum albumin, by non-covalentbinding to serum albumin via a conjugated fatty acid chain to PAM, via fusion with IgG Fc regions or transferrin, via post-translational modification attaching natural or syntheticpolymers, wherein the natural or synthetic polymer to be used is particularly HAP, moreparticularly ELP, more particularly PAS, more particularly PSA, more particularly GLK, moreparticularly XTEN and even more particularly PEG.Modified PAM may also refer to recombinant PAM (SEQ ID No.: 17) 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.In certain embodiments of the present invention, modified PAM, or a pharmaceuticalcomposition comprising modified PAM, may be administered orally, epicutaneously,subcutaneously, intradermally, transdermally, sublingually, intramuscularly, intraarterially, intravenously, or via the central nervous system (CNS, intracerebrally,intracerebroventricularly, intrathecally) or via nasal or intraperitoneal administration. Particularroutes of administration in the present invention are epicutaneous, subcutaneous, intradermal,intramuscular, intraperitoneal and intravenous, more particularly epicutaneous, subcutaneous,intradermal, intramuscular and intraperitoneal, even more particularly subcutaneous, intramuscular and intraperitoneal.The in vivo half-life of modified and / or unmodified PAM, as used herein is in particular thetime after administration to a subject upon which the level of (modified) PAM, particularly thealpha-amidating activity of the (modified) PAM is reduced by half after reaching the maximumfollowing administration. In vivo half-life time of modified PAM can for instance be determinedby administration, e.g. by injection, of modified PAM, into a test subject, e.g. an animal, determining PAM amidating activity as described herein in samples taken before administrationand at different time points post-administration, plotting the results and determining the half-life time from the resulting curve; in particular with a method analogous to the methoddescribed in Example 3. These methods can be adapted to other modified PAM types by routineadjustments. Increased half-life time of modified PAM in a specific embodiment means an increase in thehalf-life time of at least double (i.e. 2-fold), more particularly of at least 5 times, moreparticularly of at least 10 times, more particularly of at least 20 times, more particularly of atleast 50 times, more particularly of at least 100 times, even more particularly of at least 125times, more particularly of at least 150 times when compared to the half-life time of unmodifiedPAM, measured with a suitable assay, e.g. an assay as described in Example 3, after reachingits maximal value, in circulation. In another specific embodiment, increased half-life time ofmodified PAM means an increase in the half-life time of 1,05 to 2-fold, or of 1,05 to 5-fold, orof 1,05 to 10-fold, or of 1,05 to 20-fold, or of 1,05 to 50-fold, or of 1,05 to 100-fold, or of 1,05 to 125-fold, or of 1,05 to 150-fold, when compared to the half-life time of unmodified PAM, measured with a suitable assay, e.g. an assay as described in Example 3, after reaching its maximal value, in circulation In certain embodiments, the level of PAM is increased in a sample obtained from a subject having received at least one dose of the modified PAM, compared to a pre-dose sample obtained from said subject before having received said at least one dose of said modified PAM, wherein said sample is obtained from said subject 2 hours, particularly 5 hours, more particularly 10 hours, more particularly 24 hours, more particularly 48 hours, more particularly 72 hours, moreparticularly 96 hours, more particularly 120 hours, more particularly 144 hours, moreparticularly 168 hours after administration of the last dose of said modified PAM to said subject, alternatively wherein said sample is obtained from said subject 2 hours, particularly 2 to 5 hours, more particularly 2 to 10 hours, more particularly 2 to 24 hours, more particularly 2 to 48 hours, more particularly 2 to 72 hours, more particularly 2 to 96 hours, more particularly 2 to 120 hours, moreparticularly 2 to 144 hours, more particularly 2 to 168 hours after administration of the last dose of saidmodified PAM to said subject.“Bioavailability” means in particular the level, and in particular includes an elevated level, of acompound (e.g. a peptide) that is present, and in particular detectable, in the circulation of asubject over a certain period of time after administration of PAM, in particular modified PAMto said subject. Increased bioavailability means in particular a prolonged elevation of the levelof the respective compound in the subject’s circulation, after administration of said of PAM, inparticular modified PAM to said subject. Prolonged elevation of the levels of such compoundsin the subject’s circulation in particular embodiments means an elevation for at least 1 hour,more particularly for at least 8 hours, more particularly for at least 12 hours, more particularlyfor at least 24 hours, more particularly for at least 48 hours, even more particularly for at least 72 hours after administration, in particular embodiments 1 to 12 hours, or 1 to 24 hours, or 1 to 48 hours, or 1 to 72 hours after administration.As used herein, when effects of modification of PAM, such as on its half-life time in circulation,are described, they refer to the effects and changes in comparison to unmodified PAM, in particular unmodified PAM of the same type, e.g. the same isoform.Modified PAM comprises in particular embodiments a modification selected from the groupcomprising:^ Amino acid manipulation (commonly also referred to as site directed mutagenesis),including in particular the insertion, deletion and / or exchange of one or more amino acidwithin the amino acid sequence of PAM, particularly in order to reduce immunogenicityand / or proteolytic instability in vivo, compared to unmodified PAM. Thereby, bymanipulation of one or more amino acids in particular embodiments modified PAM havingenhanced protease resistance in vivo, compared to unmodified PAM, is obtained. Forexample, this involves increasing conformational stability of PAM and / or eliminating protease cleavage sites within PAM.^ Conjugation of PAM with a serum protein, such as albumin or an immunoglobulin or partsof an immunoglobulin to produce a fusion protein, wherein the conjugation in is particular embodiments either N-terminally or C-terminally, i.e. the serum protein is conjugated at the N-terminus or C-terminus of PAM. Conjugation is in particular selected from the group comprising post-translational chemical conjugation or conjugation by genetic engineering techniques. In particular, post-translational chemical conjugation includes conjugation bycrosslinking agents, such as glutaraldehyde, carbodiimides or maleimides or via aconjugated fatty acid chain to PAM. In particular, conjugation by genetic engineeringincludes conjugation by modifying a DNA sequence encoding for PAM to incorporate thegene encoding for the serum protein or parts of it; in particular embodiments, the suchcreated DNA encoding for the fusion protein encompassing PAM and the serum protein can be expressed by methods known to the skilled person to obtain the fusion protein. oFusion of PAM protein with albumin, e.g. serum albumin from a natural source orrecombinant serum albumin; oFusion of PAM with IgG Fc regions, in particular chosen from the group comprisinghuman IgG1-Fc, IgG2-Fc, IgG3-Fc, IgG4-Fc, murine IgG1-Fc, IgG2a-Fc, IgG2b-Fc, IgG3-Fc, rat IgG1-Fc, IgG2a-Fc, IgG2b-Fc, IgG2c-Fc, rabbit IgG-Fc and CanineIgG-Fc, more particularly human IgG1-Fc, IgG2-Fc, IgG3-Fc, IgG4-Fc;o Fusion of PAM with transferrin.^ Post-translational modification by attaching one or more natural or synthetic polymers toPAM, in particular polymers selected from the group comprising:o PEG (polyethylene glycol), particularly linear or branched (e.g. including 1+nbranches) PEG, particularly PEG having an average molecular weight (Mn) in therange of from 0.2 to 100 kDa, particularly 1 to 90 kDa, more particularly 2 to 80 kDa, more particularly 3 to 70 kDa, more particularly 4 to 60 kDa and even moreparticularly 5 to 50kDa, more particularly those selected form the group comprisingPEG5000, PEG10000, PEG20000 and PEG40000, wherein the number refers to theaverage molecular weight in Da. Particularly, said PEG is covalently attached toPAM. In a particular embodiment, said PEG is 5000 Da NHS-PEG is covalentlyfused to PAM. The molecular weight of PEG is determinable by well-knownmethods, such as gel permeation chromatography (aka size exclusion chromatography), or mass spectrometry, and is typically indicated by the manufacturer. oXTEN, an unstructured polypeptide, covalently fused to PAM. In a particularembodiment, XTEN is a 864 single amino acid sequence composed of amino acidsAla, Glu, Gly, Pro, Ser and Thr in a randomized manner. In certain embodiments, the half-life of a PAM-XTEN-fusion protein may be tailored by shortening of the XTENsequence. oPAS, which is a peptide polymer consisting of the amino acids proline, alanine andserine comprising 100-200, particularly 120-180, more particularly 130-170, evenmore particularly about 150 PAS repeats.o ELP (Elastin-like polypeptides) consisting of Valin-Prolin-Glycine-x-Glycinerepeats (which are naturally found in elastin), wherein x is any amino acid except forProline; in particular embodiments, the ELP comprises from 150, 200, 250, 300, 400,or 500 to 550, 600, 750, 850, 950 or 1,000 amino acids; oHAP, which is a repeated sequence of glycine rich (Gly4Ser)n polypeptide, whereinn is 100-200, particularly 120-180, particularly 130-170, particularly about 150; oGLK (gelatin-like fusion protein), a (Gly-X-Y)n structure, wherein X and Yindividually are any amino acid except for Cysteine, and with n = 60 to 1500,particularly 100 to 1250, particularly 150 to 1000, particularly 250 to 750, particularly 400 to 600, particularly about 500;o Polysaccharides, either branched or linear, which can be attached to the N-terminus,and / or C-terminus, and / or amino acid side chains (in particular of lysine) of PAM, and which in particular can be attached to PAM e.g. through N-Glycosylation, e.g.in vitro or in cell culture by selecting an appropriate expression host, and are inparticular chosen from the group comprising: ^Dextrans, in particular dextrans selected from the group comprising Dextran40, Dextran 70, and Dextran 500, wherein the number stands for the approximate molecular weight of the dextran in kilodaltons (kDa); the molecular weight of dextran is determinable by well-known methods, such as gel permeation chromatography (aka size exclusion chromatography), or mass spectrometry, and is typically indicated by the manufacturer; ^Hydroxyethyls (HES), in particular selected from the group comprisinghydroxyethylamine, hydroxyethyl methacrylate, hydroxyethyl hydrazine, hydroxyethyl disulfide and hydroxyethyl phosphonate.^ Heparosan (HEP);^ Hyaluronic acid (HA).o PSA (Polysialic acid).As used herein, “conjugation” shall in particular mean the covalent attachment of a compound or substance to the PAM protein.In certain embodiments, modification of PAM with PEG (PEGylation) shall mean theattachment of PEG as detailed herein to lysine side chains, in particular in alkaline aqueoussolution, e.g. in a buffered solution, particularly at a pH from 7.75 to 9.25, more particularlyfrom 8.25 to 8.75, more particularly about 8.5; in particular with a molar excess of PEG polymervs. PAM of 70- to 140-fold, more particularly 80- to 130-fold, more particularly 90- to 120-fold; in a particular embodiment with a method analogous to the method described in Example5, which can be adapted to other PAM types and / or other PEG polymers by routine methods. The PEGylation rate and molecular weight of the resulting PEGylated PAM can be analyzed by various well-known methods, such as gel filtration or SDS-PAGE. In other more preferred embodiments, modification of PAM is modification with PEG(PEGylation), preferably PEG with an average molecular weight of 5-10 kDa (PEG 5000 toPEG 10.000) or, more preferably with an average molecular weight of about 5 kDa (PEG 5000).In further embodiments, modification of PAM with PEG (PEGylation) shall mean theattachment of PEG 5000 or 10000 as detailed herein to serine or lysine side chains of PAM, more preferably the attachment of PEG 10000 as detailed herein to lysine side chains of PAM, or more preferably the attachment of PEG 5000 as detailed herein to lysine or serine, even more preferably to serine side chains of PAM. In other more preferred embodiments, modification of PAM is modification with XTEN as defined herein, preferably with XTEN having a sequence of SEQ ID No: 19, more preferablyto lysine or serine side chains of PAM, more preferably to lysine side chains of PAM, oralternatively via a Cys amino acid C-terminally added to PAM.PEG-PAM may refer to recombinant PAM (SEQ ID No.: 17) modified with 1+n molecules of polyethylene glycol (PEG), whereas n is an integer in the range of 0 to 100 and one PEG molecule has a molecular weight in the range of 1-100 kDa and is either a linear molecule or a branched molecule with b+1 branches, whereas b is an integer in the range of 0 to 20. In a specific embodiment PEG-PAM may refer to recombinant PAM (SEQ ID No.: 17) modified with PEG-5000 (5 kDa PEG), or PEG-10000 (10 kDa PEG). PAM modified via site directed mutagenesis may refer to recombinant PAM (SEQ ID No.: 17) with an insertion of n+1 additional amino-acids and / or n+1 deletions of amino-acids and / or n+1 exchanged amino-acids in the given amino-acid sequence (SEQ ID No.: 17), wherein n is an integer in the range of 0-100. PAM modified via fusion to native serum Albumin or recombinant human serum Albumin may refer to recombinant PAM (SEQ ID No.: 17) fused to native human serum Albumin or recombinant human serum Albumin at the N-terminus or at the C-terminus of PAM according to (SEQ ID No.: 17). PAM modified via fusion to IgG Fc regions may refer to recombinant PAM (SEQ ID No.: 17) fused to human IgG1 Fc Region at the N-terminus or at the C-terminus of PAM according to (SEQ ID No.: 17). PAM modified via XTEN may refer to recombinant PAM (SEQ ID No.: 17) fused to a XTEN moiety at the N-terminus or at the C-terminus of PAM according to (SEQ ID No.: 17) and / or to any surface exposed amino-acid of PAM according to (SEQ ID No.: 17). PAM modified via XTEN may refer to recombinant PAM (SEQ ID No.: 17) fused to XTEN moiety (SEQ ID No.: 19) at the N-terminus or at the C-terminus of PAM according to (SEQ ID No.: 17) and / or to any surface exposed amino-acid of PAM according to (SEQ ID No.: 17). In certain embodiments, site-directed mutagenesis includes creating an expression vector encoding the modified protein sequence on a genetic level to change the biochemical andbiophysical properties of PAM. Particularly, it shall mean the targeted exchange of one or moreamino acids within the region from amino acids 30 and 817 of SEQ ID No.1 with a differentamino acid.As used herein, the term "medical condition(s)" includes diseases, disorders and adverse events,more particularly the diseases, disorders and adverse events detailed herein.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 thevascular endothelium in general, BBB in particular is disrupted in several pathologicalconditions, 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, 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). Such as in vascular endothelium in general, amidated peptide hormones are regulators of the BBB in particular. Evidence is present showing that amidated hormones, such as but not limited to, bio-ADM, Oxytocin, Vasoactive intestinal Peptide (VIP), mediate the BBB function and regulation and / or have beneficial effects on BBB regeneration during and / or after conditions involving BBB disruption (Momenabadi, S. et al. Neuromolecular Med. 22, 557–571 (2020) and Yang, Jet al., J. Stroke Cerebrovasc. Dis.31, 106160 (2022)). Additionally, administration of PACAP slowed down AD-like pathology in mice and administration of VIP decreased amyloid-ß plaques formation and preserved against brain atrophy in 5xFAD transgenic mice (Korkmaz, O. T. et al. J. Mol. Neurosci.68, 389–396 (2019) and Rat, D. et al. FASEB J. 25, 3208 (2011)).In one embodiment of the present invention, circulating bio-ADM levels, but also levels ofother amidated peptides, such as, but not limited to, VIP and PACAP, are increased in clinical conditions as described above, to facilitate among others the regeneration of the endothelial barrier and / or prevent damage of endothelial barrier. Little is known about the role or influence of PAM in clinical conditions. The presence of alpha- amidating activity in human circulation was initially proved by Wand et al. (Wand, G. S., Ney, R. L., Mains, R. E. & Eipper, B. A. Characterization of peptide alpha-amidation activity in human cerebrospinal fluid and central nervous system tissue.et al., Neuroendocrinology 41, 482–489 (1985)). They reported no sex differences but some variations of PAM activity in certain disease states: Plasma PAM activities were increased in hypothyroid adults as well as in patients with medullary thyroid carcinoma. The activity of PAM in tissues of medullary thyroid carcinoma, pheochromocytoma and pancreatic islet tumors were shown to be elevatedsuggesting increased formation of amidated peptides in endocrine tumor tissues (Gether, U.,Aakerlund, L. & Schwartz, T. W. Comparison of peptidyl-glycine α-amidation activity in medullary thyroid carcinoma cells, pheochromocytomas, and serum. et al., Mol. Cell. Endocrinol. 79, 53–63 (1991) and Wand, G. S., Nev, R. L., Mains, R. E. & Eipper, B. A. Characterization of Peptide Alpha-Amidation Activity in Human Cerebrospinal Fluid and Central Nervous System Tissue, et al., Neuroendocronology 41, 482–489 (1985)). Patients suffering from multiple endocrine neoplasia type 1 (MEN-1) and pernicious anemia showed a decreased plasma PAM activity in comparison to healthy control subjects. The presence of amidating activity in human cerebrospinal fluid (CSF) was shown by Wand and colleagues. In patients suffering from Alzheimer’s disease (AD) plasma PAM activities were shown to be unaltered when compared to healthy controls, while CSF PAM activities were significantly decreased in comparison to activities from normal specimen (Wand, G. S. et al. Alzheimer’s disease: Low levels of peptide a-amidation activity in brain and CSF. Neurology 37, 1057–1061 (1987)). In addition, in WO2015 / 103594 it was proposed that the presence of PAM-Protein in CSF detected by mass spectrometry of AD-patients was reduced compared to healthy controls. Moreover, bio-ADM, one of the hormones amidated by PAM, was shown to be reduced in patients with prevalent and incident Alzheimer’s disease (WO2019 / 154900). Direct associations of circulating PAM activities were reported to be associated with prediction, diagnosis or progression of AD in WO2021170816A1 and WO2021170752A1. PAM activities of patients suffering from multiple sclerosis (MS) were shown to be increased in CSF, with a significant decrease in serum (Tsukamoto, T. et al. Increased Peptidylglycine alpha-Amidating Monooxygenase Activity in Cerebrospinal Fluid of Patients with Multiple Sclerosis. Intern. Med. 34, 229–232 (1995)) and WO2010 / 005387). An association between plasma activity of PAM and type-2 -diabetes was described in (WO2014 / 118634). Further, WO2021170816A1 and WO2021170752A1 show an increase of PAM activity in circulation due to acute conditions such as sepsis and shock. So far, no clinical testing of PAM as a drug candidate has been reported. 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 indispensablefor an intact endothelial and blood-brain barrier function and that administration of bio-ADM and otherpeptide hormones to supra-physiological levels has protective effects towards endothelial and blood-brain barrier function (for review Kis B et al.,. Peptides. 2006 Jan;27(1):211-22) The BBB protectsneurons 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, M. D. et al., Nat. Rev. Neurol. 14, 133–150 (2018)). BBB dysfunction occurs in severaldiseases, including MS, epilepsy, stroke and dementia, including Alzheimer’s disease (AD). In theseconditions, BBB dysfunction plays a central role in the pathology, as BBB disruption leads to iondysregulation, edema, neuroinflammation, culminating in neuronal dysfunction, increased intracranial pressure and neuronal degeneration (Profaci, C. P. et al., Experimental Medicine, 217.4 (2020)). The pharmaceutical composition according to the present invention is capable of treating and / or preventing BBB dysfunction and dysregulation 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 cognitiveimpairment, dementia, cerebrovascular dementia, Alzheimer's disease, and encephalitis.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(1)). 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 producedby and / or acting on the endothelium (Deanfield J et al., J Hypertens. 2005 Jan;23(1):7-17.) Normalfunctions of endothelial cells include mediation of coagulation, platelet adhesion, immune function andcontrol 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 totransition between quiescent and activated states (Colombo PC et al., Curr Heart Fail Rep. 2015Jun;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 (Gutiérrez E, et al., Eur Heart J. 2013 Nov;34(41):3175-81)). 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 weightsolutes (Rocker GM, et al., Thorax. 1987 Aug;42(8):620-3).). Endothelial dysfunction can result fromand / or contribute to several disease processes, as occurs in hypertension, hypercholesterolaemia, diabetes or septic shock. Endothelial dysfunction is a major pathophysiological mechanism that leadstowards coronary artery disease, and other atherosclerotic diseases. Therefore, the pharmaceuticalcomposition of this invention may be used for treatment and / or prevention of diseases associated with an endothelial and / or vascular dysfunction and / or disruption. Therefore, the shared hallmark of the diseases described above and listed below is a dysfunction of the endothelial barrier, including, but not limited to, the vascular endothelium, the blood-brain barrier andthe endothelial barrier of the intestinal tract. The symptoms, diseases and / or disorders include, but arenot limited to, the following:(1) Brain and neurological diseases: cerebral infarction, mild cognitive impairment, dementia,cerebrovascular dementia, Alzheimer's disease, and encephalitis. (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. (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. (4) Endocrine and metabolic disorders: diabetes and diabetic organ disorders(e.g., diabetic nephropathy or diabetic retinopathy). (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.(6) Brain / neurological disorders prevented or treated by the pharmaceutical composition according toan embodiment of the present invention is dementia, in particular mild cognitive impairment, cerebralinfarction dementia or Alzheimer’s disease. 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) ofdementia 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). 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 (Aβ1-42), dimers, trimers, oligomers and subsequent amyloid aggregation and deposition in plaques, abnormal hyperphosphorylation and aggregation of tau protein, progressive intracellular neurofibrillarydegeneration, changes within the innate immune system and inflammation and a breakdown of theblood-brain barrier. 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 MCIpatients adds evidence to the hypothesis of BBB breakdown to precede neurodegeneration. 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 withBBB dysfunction and microvascular lesions (Janelidze S et al., Neurobiol Aging. 2017 Mar;51:104-112.). Additional symptoms may include fluctuations in alertness, visual hallucinations, slowness ofmovement, 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 DLBexists. For review see McKeith et al. 2017. Neurology 89: 88-100.Vascular dementia (VaD), also known as multi-infarct dementia (MID) and vascular cognitive impairment (VCI), is dementia caused by problems in the supply of blood to the brain, typically a series of minor strokes, leading to worsening cognitive decline that occurs step by step. The term refers to a syndrome consisting of a complex interaction of cerebrovascular disease and risk factors that lead tochanges in the brain structures due to strokes and lesions and resulting in disruption of the blood-brainbarrier and changes in cognition. The temporal relationship between a stroke and cognitive deficits isneeded to make the diagnosis. 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, andtypical loss of over 70% of spindle neurons, while other neuron types remain intact. FTD is associatedwith BBB dysfunction and microvascular lesions (Janelidze S, et al., Neurobiol Aging. 2017Mar;51:104-112).and accounts for 20% of young-onset dementia cases. Signs and symptoms typicallymanifest 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 multiplecerebrovascular events (strokes). For review see Venkat et al. 2015. Exp Neurol 272: 97–108.Nevertheless, a pathology that is common for all forms of dementia is an impairment of the blood-brain barrier.As used herein, “dementia " refers to substantially the clinical syndrome characterized by a cluster ofsymptoms and signs manifested by difficulties in memory, disturbances in language, psychological andpsychiatric changes, and impairments in activities of daily living as outlined above have a commondenominator 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.(7) Infectious diseases prevented or treated by the pharmaceutical composition according to anembodiment of the present invention are, in particular, sepsis or septic shock.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 1;117(1):60-73). Hemodynamic instability plays animportant 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., Shock. 2018 Dec;50(6):648-654.). As shown inexample 8, the pharmaceutical composition according to the present invention may be used forprotecting the endothelial barrier from dysregulation and / or prevent endothelial permeability.(8) The gastrointestinal diseases prevented or treated by the pharmaceutical composition according toan embodiment of the present invention are, in particular, inflammatory diseases (e.g., inflammatorybowel disease (IBD) or Crohn's disease), ulcerative diseases (e.g., ulcerative colitis) or intestinal Bechet's disease. Inflammatory bowel diseases (IBD) include Crohn’s disease (CD), ulcerative colitis (UC) (and indeterminate colitis), which share several inflammatory characteristics with other chronic immunedisturbances including immune activation, leukocyte infiltration into tissues and increased vasculardensity. The maintenance of normal vascular barrier supports nutrient and O2 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., 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 8. (9) 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). Glucagon-like peptide-1 (GLP-1)-based therapy of type 2 diabetes is executed either by GLP-1 receptor agonists, such as Liraglutide, Albiglutide or Taspoglutide, which stimulate the GLP-1 receptors, or by dipeptidyl peptidase-4 (DPP-4) inhibitors, which prevent the inactivation of endogenous GLP-1 thereby increasing the concentration of endogenous active GLP-1. GLP-1 and its analogues activate pancreatic receptors resulting in improved glycemia through glucose-dependent stimulation of insulin secretion and inhibition of glucagon secretion. There is also a potential beta cell preservation effect, as judged from rodent studies. GLP-1 receptors are additionally expressed in extrapancreatic tissue, havingpotential for the treatment to reduce body weight and to potentially have beneficial cardio- andendothelioprotective effects. Clinical trials in subjects with type 2 diabetes have shown that in periods of 12 weeks or more, GLP-1 analogues treatments are efficient both as monotherapy and in combination therapy with metformin, sulfonylureas, thiazolidinediones or insulin. Furthermore, GLP-1 receptor agonists reduce body weight, whereas DPP-4 inhibitors are body weight neutral. The treatment is safe with very low risk for adverse events, including hypoglycemia. GLP-1 based therapy is now well established therapy of type 2 diabetes, with a particular value in combination with metformin in patients who are inadequately controlled by metformin alone (Ahrén B. Exp Cell Res.2011 May 15;317(9):1239-45). GLP-1 is a peptide-hormone in need of amidation by PAM.The pharmaceutical composition according to the present invention is a medication that is preferablyused 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).In particular embodiments, the pharmaceutical composition according to the present invention is amedication 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 andgastrointestinal disorders) and comprises in addition Vitamin C. Likewise, the embodiments of theinvention relating to a compound, combination or method of treatment and / or prevention, include in particular embodiments the addition of Vitamin C.In embodiments of the present invention, the pharmaceutical composition is used to treat and / or preventcardiovascular 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.In preferred embodiments of the present invention, the pharmaceutical composition is used to treatand / or prevent heart failure, pulmonary hypertension, hypertension, organ damage due to hypertension, peripheral vascular disease, and arteriosclerosis.In most preferred embodiments of the present invention, the pharmaceutical composition is used to treatand / or pulmonary hypertension and hypertension.In other embodiments of the present invention, the pharmaceutical composition is used to treat and / orprevent endocrine and metabolic disorders such as diabetes and diabetic organ disorders (e.g., diabetic nephropathy or diabetic retinopathy).In preferred embodiments of the present invention, the pharmaceutical composition is used to treatand / or prevent type-2 diabetes.In further embodiments of the present invention, the pharmaceutical composition is used to treat and / orprevent 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.In preferred embodiments of the present invention, the pharmaceutical composition is used to treatand / 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.In most preferred embodiments of the present invention, the pharmaceutical composition is used to treatand / or prevent inflammatory bowel disease and / or Crohn's disease.In other embodiments of the present invention, the pharmaceutical composition is used to treat and / orprevent 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.In preferred embodiments of the present invention, the pharmaceutical composition is used to treatand / or prevent sepsis and / or septic shock.In embodiments of the present invention, the pharmaceutical composition is used to treat and / or preventbrain and neurological diseases such as cerebral infarction, mild cognitive impairment, dementia, cerebrovascular dementia, Alzheimer's disease, and encephalitis.In preferred embodiments of the present invention, the pharmaceutical composition is used to treatand / or prevent cerebral infarction.In more preferred embodiments of the present invention, the pharmaceutical composition is used to treatand / or prevent dementias, including mild cognitive impairment, cerebrovascular dementia, and Alzheimer’s disease.In most preferred embodiments of the present invention, the pharmaceutical composition of the presentinvention is used for the prevention and / or treatment of symptoms associated with mild cognitive impairment (MCI) and Alzheimer’s Disease (AD).In certain embodiments of the present invention, the pharmaceutical composition is used to treat and / orprevent pathological disorders associated with endothelial and / or blood-brain barrier disorders.In preferred embodiments of the present invention, the pharmaceutical composition is used to treatand / or endothelial and / or blood-brain barrier disfunction associated with sepsis or septic shock. Certain embodiments of the present invention relate to modified PAM for use in treatment or prevention of a disease or medical condition in a subject, wherein said disease or medical condition is associated with endothelial barrier dysfunction and / or blood-brain barrier dysfunction. Further preferred embodiments of the present invention relate to modified PAM for use in treatment or prevention of a disease in a subject, wherein said disease or medical condition is selected from the group consisting of cardiovascular, kidney, inflammatory, infectious, and metabolic diseases or disorders, dementia, cancer, and other diseases or medical conditions connected with disturbed peptide homeostasis, wherein more particularly said disease or disorder is sepsis.In more preferred embodiments of the present invention, the pharmaceutical composition is used to treatand / or prevent endothelial and / or blood-brain barrier dysfunction associated with cerebral infarction.In more preferred embodiments of the present invention, the pharmaceutical composition is used to treatand / or prevent endothelial and / or blood-brain barrier dysfunction associated with dementias, including mild cognitive impairment, cerebrovascular dementia, and Alzheimer’s disease.In most preferred embodiments of the present invention, the pharmaceutical composition is used to treatand / or prevent endothelial and / or blood-brain barrier dysfunction associated with mild cognitive impairment (MCI) and Alzheimer’s Disease (AD).More preferably, the pharmaceutical composition according to the present invention is used for theprevention 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, diabeticretinopathy, pulmonary fibrosis, sepsis, or septic shock. 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). The term “amino acids” as used herein in particular refers to naturally occurring amino acids, more particularly amino acids naturally occurring in animals, more particularly amino acids naturally occurring in mammals, more particularly the 20 canonical amino acids. In certain embodiments, the medical condition is a disease or disorder characterized by disturbed peptide homeostasis characterized by, in a sample of bodily fluid or a tissue extract obtained from a subject, ^the level of PAM and / or its isoforms and / or fragments, in particular proteins comprisingan amino acid sequence of SEQ ID. No 1 to 10, is predetermined, particularly below 24Units / L, more particularly below 20 Units / L, more particularly below 16 Units / L, moreparticularly below 14 Units / L and most particularly below 10 Units / L and / or^ the ratio peptide-Gly / peptide-amide of one or more peptides is above a predeterminedthreshold, wherein particularly the ratio is higher than 1, more particularly 1.5 or higher,more particularly 1.6 or higher, more particularly 1.7 or higher, more particularly 1.8 orhigher, more particularly 1.9 or higher, even more particularly 2 or higher, and whereinindependently in particular the peptides are chosen from the group comprisingAdrenomedullin, Substance P, Vasopressin, Oxytocin, Amylin, PAMP, alpha-MSH, Calcitonin, Kisspeptin, Neuropeptide Y, Vasoactive intestinal peptide and Thyroliberin. In certain embodiments, the medical condition is a disease or disorder characterized by disturbed peptide homeostasis characterized by, in a sample of bodily fluid or a tissue extract obtained from a subject,• the level of PAM and / or its isoforms and / or fragments, in particular proteins comprising anamino acid sequence of SEQ ID No.1 to 10, is the activity of PAM, is predetermined, and is particularly below 24 Units / L, more particularly below 20 Units / L, more particularly below 16 Units / L, more particularly below 14 Units / L and most particularly below 10 Units / L and / or• the level of PAM and / or its isoforms and / or fragments, in particular proteins comprising anamino acid sequence of SEQ ID No. 1-10, is the total concentration of PAM, is predetermined and is particularly below 100 ng / mL, preferably below 90 ng / mL, more preferably below 80 ng / mL, more preferably below 60 ng / mL, more preferably below 50 ng / mL and most preferably below 40 ng / mL and / or• the ratio peptide-Gly / peptide-amide of one or more peptides is above a predeterminedthreshold, wherein particularly the ratio is higher than 1, more particularly 1.5 or higher, more particularly 1.6 or higher, more particularly 1.7 or higher, more particularly 1.8 or higher, more particularly 1.9 or higher, even more particularly 2 or higher, and wherein independently in particular the peptides are chosen from the group comprising Adrenomedullin, Substance P, Vasopressin, Oxytocin, Amylin, PAMP, alpha-MSH, Calcitonin, Kisspeptin, Neuropeptide Y, Vasoactive intestinal peptide and Thyroliberin.In particular embodiments, the disease or disorder with disturbed peptide homeostasis ischaracterized by, in a sample of bodily fluid or tissue extract obtained from a subject, ^the ratio of ADM-Gly / bio-ADM is above a predetermined threshold, in particularhigher then 1, more particularly 1.5 or higher, more particularly 1.6 or higher, moreparticularly 1.7 or higher, more particularly 1.8 or higher, more particularly 1.9 orhigher, even more particularly 2 or higher and / or^ the bio-ADM concentration is below a predetermined threshold, in particular below 20pg / mL, more particularly below 15 pg / mL, more particularly below 10 pg / mL, moreparticularly below 5 pg / mL.In one embodiment, said -molar ratio of said Peptide-Gly and its corresponding Peptide-amide(Peptide-NH2) (Peptide-Gly / Peptide-amide) and / or -concentration of PAM and / or- activity of PAMin a bodily fluid of the subject may be determined at least once before and / or at least once after the treatment of the subject. In a more specific embodiment, said -molar ratio of ADM-Gly and bio-ADM (ADM-Gly / bio-ADM) and / or- concentration of bio-ADM and / or- concentration of PAM and / or- activity of PAMin a bodily fluid of the subject may be determined at least once before and / or at least once after the treatment of the subject.A person skilled in the art will understand, that the aforementioned concentration of PAM oractivity of PAM comprises endogenous PAM and / or its isoforms and / or fragments thereof, aswell as exogenous (therapeutically applied) PAM and / or its isoforms and / or fragments thereofincluding recombinant PAM according to SEQ ID No.: 17 either modified or unmodified, aftersaid subject has been treated with said PAM.In certain embodiments, the threshold is predetermined by measuring, such as the level of PAMand / or its isoforms and / or fragments and / or ADM-Gly and / or bio-ADM, in samples obtainedfrom a cohort of subjects and calculating e.g. the 25th percentile, more particularly the 10thpercentile, even more particularly the 5th percentile to define the threshold by which subjectsare characterized as suffering from a medical condition or as being at risk of getting a medical condition.As used herein, the cohort of subjects may refer to a representative sample of subjects from thegeneral population, which is typically randomly chosen; in certain embodiments, this may forinstance be a study population from a medical study, and in particular embodiments may relatea certain cohort of interest, e.g. in terms of a certain age range, sex, certain physical conditions,biomarkers, such as BMI, or the like. In certain particular embodiments, the cohort of subjectsconsists of apparently healthy, in particular self-reported healthy, asymptomatic subjects; someof these subjects may in fact have an underlying, hitherto undiagnosed medical condition inconnection with reduced PAM levels. In particular embodiments, the level of PAM and / or its isoforms and / or fragments thereof is detected as total PAM concentration and / or PAM activity. In particular this means the total concentration of all PAM isoforms, and the total alpha-amidating activity determinably in a sample.In other embodiments, particular thresholds for PAM activity in a sample, in particular a plasmasample, obtained from a subject are 24 Units / L, more particular 20 Units / L, more particular 16Units / L, more particular 14 Units / L and even most particular 10 Units / L and even moreparticular 5 Units / L, determined using a PAM activity assay, in particular the PAM activityassay as described herein. Particular thresholds for the bio-ADM level are 15 pg / ml, moreparticularly 10 pg / ml, even more particularly 5 pg / mL.In certain embodiments, the threshold is pre-determined by measuring the ratio of peptide-Gly / peptide-amide, in particular ADM-Gly to bio-ADM, in a cohort of subjects and calculatinge.g. the 75thpercentile, more particularly the 90thpercentile, even more particularly the 95thpercentile to define subjects suffering from a medical condition or at risk of getting a medicalcondition.In other embodiments, a particular threshold for the ratio of ADM-Gly to bio-ADM in a sampleobtained from a subject is 1, more particularly 1.5, more particularly 2, more particularly 2.5,more particularly 5, more particularly 7.5, even more particularly 10.A particular embodiment relates to PEGylated PAM for use in the treatment of a diseasecharacterized by disturbed peptide homeostasis in a subject, in particular wherein PEGylatedPAM is administered in a dose of 2.0 Units / kg to 116.1 Units / kg, particularly 2.1 Units / kg to71.5 Units / kg, more particularly 2.2 Units / kg to 54.1 Units / kg, more particularly 2.3 Units / kgto 38.1 Units / kg, more particularly 2.4 Units / kg to 24.1 Units / kg, even more particularly 2.5Units / kg to 11.1 Units / kg. It is apparent that this relates likewise to unmodified PAM for use in the treatment of a diseasecharacterized by disturbed peptide homeostasis in a subject and / or modified PAM for use in thetreatment of a disease characterized by disturbed peptide homeostasis in a subject. To clarify, particular embodiments likewise relate to unmodified PAM for use in the treatment of a disease characterized by disturbed peptide homeostasis in a subject, in particular wherein unmodified PAM is administered in a dose of 2.0 Units / kg to 116.1 Units / kg, particularly 2.1 Units / kg to 71.5 Units / kg, more particularly 2.2 Units / kg to 54.1 Units / kg, more particularly 2.3 Units / kg to 38.1 Units / kg, more particularly 2.4 Units / kg to 24.1 Units / kg, even more particularly 2.5Units / kg to 11.1 Units / kg. To clarify, particular embodiments likewise relate to modified PAMfor use in the treatment of a disease characterized by disturbed peptide homeostasis in a subject, in particular wherein modified PAM is administered in a dose of 2.0 Units / kg to 116.1 Units / kg, particularly 2.1 Units / kg to 71.5 Units / kg, more particularly 2.2 Units / kg to 54.1 Units / kg, more particularly 2.3 Units / kg to 38.1 Units / kg, more particularly 2.4 Units / kg to 24.1 Units / kg, even more particularly 2.5 Units / kg to 11.1 Units / kg.In addition, more particular embodiments relate to PEGylated PAM for use in the treatment ofa disease characterized by disturbed peptide homeostasis in a subject, in particular whereinPEGylated PAM, more particularly PEG 5000 or PEG 10000 is administered in a dose of 2.0Units / kg to 116.1 Units / kg, particularly 2.1 Units / kg to 71.5 Units / kg, more particularly 2.2Units / kg to 54.1 Units / kg, more particularly 2.3 Units / kg to 38.1 Units / kg, more particularly 2.4 Units / kg to 24.1 Units / kg, even more particularly 2.5 Units / kg to 11.1 Units / kg.In addition, more particular embodiments relate to xtenylated PAM for use in the treatment ofa disease characterized by disturbed peptide homeostasis in a subject, in particular whereinxtenylated PAM, is administered in a dose of 2.0 Units / kg to 116.1 Units / kg, particularly 2.1Units / kg to 71.5 Units / kg, more particularly 2.2 Units / kg to 54.1 Units / kg, more particularly 2.3 Units / kg to 38.1 Units / kg, more particularly 2.4 Units / kg to 24.1 Units / kg, even more particularly 2.5 Units / kg to 11.1 Units / kg. In particular embodiments, the disease or disorder characterized by disturbed peptidehomeostasis is selected from the group comprising but not limited to:^ dementia, particularly dementia selected from the group comprising mild cognitiveimpairment (MCI), Alzheimer’s disease, vascular dementia, mixed Alzheimer’s disease, and vascular dementia, Lewy body dementia, frontotemporal dementia, focal dementias (including progressive aphasia), subcortical dementias (including Parkinson’s disease) and secondary causes of dementia syndrome (including intracranial lesions); ^cardiovascular disorders, particularly cardiovascular disorders selected from the groupcomprising atherosclerosis, hypertension, heart failure (including acute and acute decompensated heart failure), atrial fibrillation, cardiovascular ischemia, cerebral ischemic injury, cardiogenic shock, stroke (including ischemic and haemorrhagic strokeand transient ischemic attack) and myocardial infarction; ^kidney diseases, particularly kidney diseases selected from the group comprising renaltoxicity (drug-induced kidney disease), acute kidney injury (AKI), chronic kidney disease (CKD), diabetic nephropathy, and end-stage renal disease (ESRD); ^infectious diseases caused by infectious organisms such as bacteria, viruses, fungi orparasites, more particularly by infectious bacteria, particularly diseases selected from thegroup comprising SIRS, sepsis, and septic shock; ^metabolic diseases, particularly metabolic diseases selected from the group comprisingdiabetes type 1, diabetes type 2, and metabolic syndrome.In embodiments of the present invention, the pharmaceutical composition comprises acombination of modified or unmodified PAM and Vitamin C.In other embodiments the pharmaceutical composition comprises a combination of modified orunmodified PAM with Vitamin C, wherein said pharmaceutical composition may be used forthe treatment and / or prevention of type-2 diabetes.In other embodiments the pharmaceutical composition comprises a combination of modified orunmodified PAM with Vitamin C, wherein said pharmaceutical composition may be used forthe treatment and / or prevention of IBD.In other embodiments the pharmaceutical composition comprises a combination of modified orunmodified PAM with Vitamin C, wherein said pharmaceutical composition may be used for thetreatment of a disease or disorder selected from the group consisting of cardiovascular, kidney, inflammatory, infectious, and metabolic diseases or disorders, dementia, cancer, and other diseases or disorders connected with disturbed peptide homeostasis, wherein more particularly said disease or disorder is sepsis.In further embodiments of the present invention a combination of modified or unmodified PAMfor use in treatment and / or prevention of cardiovascular, edematous and / or inflammatorydiseases, such as sepsis or septic shock, wherein unmodified or modified PAM is to be used incombination with Vitamin C.Further embodiments of the present invention relate to modified or unmodified PAM for use intreatment of a disease in a subject, wherein modified or unmodified PAM is to be used incombination 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.An embodiment of the present invention is modified or unmodified PAM for use in treatmentof a disease in a subject, wherein modified or unmodified PAM is to be used in combinationwith Vitamin C and wherein• preferably 1.5-54 Units / kg of PAM are combined with 1-10000 mg / kg, preferably 2-8000 mg / kg, more preferably 3-6000 mg / kg, more preferably 4-4000 mg / kg, more preferably 5-2000 mg / kg, more preferably 10-1000 mg / kg of Vitamin C,• more preferably 1.7-33 Units / kg PAM are combined with 1-10000 mg / kg, preferably 2-8000 mg / kg, more preferably 3-6000 mg / kg, more preferably 4-4000 mg / kg, more preferably 5-2000 mg / kg, more preferably 10-1000 mg / kg of Vitamin C,• more preferably 1.8-26 Units / kg PAM are combined with 1-10000 mg / kg, preferably 2-8000 mg / kg, more preferably 3-6000 mg / kg, more preferably 4-4000 mg / kg, more preferably 5-2000 mg / kg, more preferably 10-1000 mg / kg of Vitamin C,• more preferably 1.9-20.6 Units / kg PAM are combined with 1-10000 mg / kg, preferably2-8000 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,• more preferably 2-14 Units / kg of PAM are combined with 1-10000 mg / kg, preferably2-8000 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.This includes for example that Vitamin C is applied as separate injection and / or infusion andmodified or unmodified PAM is applied as separate injection and / or infusion.Moreover, Vitamin C and modified or unmodified PAM, if administered separately, may beadministered via the same or different routes of application. This means for example thatVitamin C is administered subcutaneously and modified or unmodified PAM intravenously,respectively.It is to be understood that both, Vitamin C and modified or unmodified PAM are administeredas exogenous substances.The compounds or combinations of the invention are in particular administered intramuscularlyor intraperitoneally or subcutaneously or intravenously.An embodiment of the present invention is a pharmaceutical combination or pharmaceuticalcomposition, wherein Vitamin C is to be used in combination with peptidylglycine α-amidatingmonooxygenase (PAM) wherein said PAM 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 due to a conjugated fatty acid chain to PAM, mor preferably via fusion with IgG Fc regions or Transferrin, preferably modified to act as PEG-based carrier prodrug, most preferably via post-translational modifications attachingnatural 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.Other embodiments of the present invention relate to a method for a long-lasting elevation ofamidated peptide hormones like Peptide-Gly in the circulation by the combinatory use of its glycine-extended precursors with the activator PAM and in one embodiment with the Cofactor Vitamin C, as well as the use of the method to treat and / or prevent an acute and / or chronic disease with such combinations. The PAM enzyme is present in circulation and is capable of amidation in circulation. 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 a combination of PAM and Vitamin C. Particular embodiments of the invention are:1. Modified peptidyl-glycine alpha-amidating monooxygenase (PAM) having an increased invivo half-life compared to unmodified PAM, wherein the enzymatic activity of modifiedPAM is maintained compared to unmodified PAM.2. A method for increasing the in vivo half-life of PAM, wherein said method comprisesmodifying said PAM, wherein said in vivo half-life is increased compared to unmodifiedPAM, and wherein the enzymatic activity of PAM is maintained.In particular embodiments of the invention, said in vivo half-life is increased in a subject. Inparticular embodiments, said in vivo half-life is increased in a subject having received at leastone administration of the modified PAM. The enzymatic activity of PAM is maintained means that the modified PAM has PAM enzymatic activity as further described herein. In particular embodiments, the enzymaticactivity of PAM is the in vitro or in vivo, in particular the in vivo enzymatic activity of PAM.In particular, this relates to the amidating activity of PAM as described herein.3. Modified PAM according to embodiment 1 or method according to embodiment 2, whereinPAM is a protein comprising a polypeptide sequence selected from the group comprising SEQ ID Nos 1 to 10, or having at least 85%, particularly at least 90%, more particularly at least 95%, even more particularly at least 99% sequence homology therewith, alternatively wherein sequence homology therewith means a sequence homology of 85% to 100%, preferably of 90% to 100%, or more preferably of 95 to 100% or most preferably of 99% to 100%.Modified PAM or method according to any of the preceding embodiments, wherein theenzymatic activity of PAM is the alpha-amidating activity of PAM.Modified PAM or method according to any of the preceding embodiments, wherein PAMis modified bya. attaching one or more natural or synthetic polymers units, wherein said polymers areselected from the group comprising i. PEG, particularly having an average molecular weight in the range of 0.2-100kDa, particularly 1-90 kDa, more particularly 2-80 kDa, more particularly 3-70 kDa, more particularly 4-60 kDa and even more particularly 5 to 50kDa; ii. XTEN;iii. a peptide polymer consisting of repeats of PAS (the amino acids proline, alanineand serine, particularly comprising 100-200, particularly 120-180, more particularly 130-170, even more particularly about 150 PAS repeats; iv. elastin-like polypeptides consisting of Valin-Prolin-Glycine-x-Glycine repeats,wherein x is any amino acid other than Proline, particularly consisting of from 150, 200, 250, 300, 400, or 500 to 550, 600, 750, 850, 950 or 1,000 amino acids; v. HAP polypeptide, having the sequence (Gly4Ser)n, wherein n is 100-200;vi. gelatin-like fusion protein, having the sequence (Gly-X-Y)n, wherein X and Yindividually are any amino acid except for Cysteine, and wherein n = 60 to 1500; vii. polysaccharides, in particular those chosen from the group comprising:1. dextrans;2. hydroxyethyls;3. heparosan;4. hyaluronic acid;viii. polysialic acid.b. conjugation with a serum protein, such as albumin or an immunoglobulin or parts of animmunoglobulin;c. site-directed mutagenesis by insertion, deletion and / or exchange of one or more aminoacids within the amino acid sequence of PAM.Modified PAM or method according to any of the preceding embodiments, wherein the invivo half-life of said modified PAM, compared with unmodified PAM, is increased by atleast 2-fold, particularly at least 5-fold, more particularly at least 10-fold, more particularly at least 20-fold, more particularly at least 50-fold, more particularly at least 100-fold, more particularly at least 150-fold, or alternatively 1,05 to 2-fold, or of 1,05 to 5-fold, or of 1,05 to 10-fold, or of 1,05 to 20-fold, or of 1,05 to 50-fold, or of 1,05 to 100-fold, or of 1,05 to 125-fold, or of 1,05 to 150-fold.7. Modified PAM or method according to any of the preceding embodiments, wherein thelevel of PAM is increased in a sample obtained from a subject having received at least one dose of said modified PAM, compared to a pre-dose sample obtained from said subject before having received said at least one dose of said modified PAM, wherein said sample is obtained from said subject 2 hours, particularly 5 hours, more particularly 10 hours, more particularly 24 hours, more particularly 48 hours, more particularly 72 hours, more particularly 96 hours, more particularly 120 hours, more particularly 144 hours, more particularly 168 hours after administration of the last dose of said modified PAM to said subject, alternatively wherein said sample is obtained from said subject 2 hours, particularly 2 to 5 hours, more particularly 2 to 10 hours, more particularly 2 to 24 hours, more particularly 2 to 48 hours, more particularly 2 to 72 hours, more particularly 2 to 96 hours, more particularly 2 to 120 hours, more particularly 2 to 144 hours, more particularly 2 to 168 hours after administration of the last dose of said modified PAM to said subject. In particular embodiments, the level of PAM is increased relates to the increase of the total level of PAM including modified PAM and unmodified PAM (wherein said unmodified PAM is naturally present in a subject).8. Modified PAM or method according to any of the preceding embodiments wherein theincrease of PAM level and or increase of PAM half-life is determined in a sample obtained from a subject having received at least one dose of said modified PAM, by determining the alpha-amidating activity and / or the level of amidated peptide hormones and / or by determining the PAM concentration in said sample.9. Modified PAM or method according to any of the preceding embodiments, wherein the invivo half-life of said modified PAM, compared with unmodified PAM, is increased by atleast 0.5 hours, more particularly at least 1 hour, more particularly at least 2 hours, more particularly at least 5 hours, more particularly at least 10 hours, more particularly at least24 hours, more particularly at least 48 hours, more particularly at least 60 hours,alternatively by 0.5 to 1 hour, more particularly 0.5 to 2 hours, more particularly 0.5 to 5 hours, more particularly 0.50 to 10 hours, more particularly 0.5 to 24 hours, more particularly 0.5 to 48 hours, more particularly 0.5 to 60 hours.Modified PAM or method according to any of the preceding embodiments, wherein the invivo half-life of said modified PAM is at least 1 hour, more particularly 2 hours, moreparticularly at least 5 hours, more particularly at least 10 hours, more particularly at least 24 hours, more particularly at least 48 hours, more particularly at least 60 hours, alternatively 0.5 to 1 hour, more particularly 0.5 to 2 hours, more particularly 0.5 to 5 hours, more particularly 0.50 to 10 hours, more particularly 0.5 to 24 hours, more particularly 0.5 to 48 hours, more particularly 0.5 to 60 hours.Modified PAM or method according to any of the preceding embodiments, wherein the invivo half-life is the half-life level in the blood circulation of a subject.Modified PAM or method according to any of the preceding embodiments, wherein at least50%, particularly at least 75%, particularly at least 85%, particularly at least 95%, particularly about 100% of the enzymatic activity is maintained, compared to unmodified PAM, alternatively wherein enzymatic activity is maintained means an enzymatic activity from 50% to 100%, preferably from 75% to 100%, more preferably from 85% to 100%,more preferably from 95% to 100% or most preferably 100% compared to unmodifiedPAM.Modified PAM or method according to any of the preceding embodiments, wherein saidmodified PAM, when administered to a subject, increases the bioavailability of one or more amidated peptides, in particular one or more peptide hormones in said subject.Pharmaceutical composition comprising a modified PAM according to any of embodiments1 to 13.Pharmaceutical composition according to embodiment 14, comprising one or morepharmaceutically acceptable excipients, such as surfactants, emulsifiers, or carriers.16. Pharmaceutical composition according to embodiment 14 or 15, which is a ready-to-useformulation.17. Pharmaceutical composition according to any of embodiments 14 to 16, which is a freeze-dried composition. In certain embodiments, the pharmaceutical composition according to the invention, comprises one or more items from the group comprising packaging, instructions to the subject and / or physician treating the subject and a leaflet. Such items may in particular comprise instructions regarding the administration of the pharmaceutical composition to a subject, such as relating to the dosage, medical indications, administration route,18. Modified PAM according to any of embodiments 1 to 13 or pharmaceutical compositionaccording to any of embodiments 14 to 17 for use as a medicament. Certain embodiments of the present invention relate to the use of a modified PAM apharmaceutical composition according to the present invention for the manufacture of amedicament. Certain embodiments of the present invention relate to the use of a modified PAM a pharmaceutical composition according to the present invention for the treatment of medical conditions. Certain embodiments of the present invention relate to a method of treatment of a subject in need thereof, said method comprising administering to said subject a therapeutically effectiveamount of a modified PAM a pharmaceutical composition according to the present invention.Specific embodiments of the present invention relating to the modified PAM or pharmaceutical composition for use as a medicament, i.e. embodiments relating to the modified PAM or pharmaceutical composition for use in treatment or prevention of certain medical conditions or for reducing the risk of a subject for suffering from a medical condition, analogously read on the aforementioned use for the manufacture of a medicament, use for the treatment of medical conditions and / or method of treatment.19. Modified PAM according to any of embodiments 1 to 13 or pharmaceutical compositionaccording to any of embodiments 14 to 17 for reducing the risk of a subject for suffering from a medical condition, and / or for reducing the occurrence of a disease or disorder in said subject, and / or for reducing the severity of a disease or disorder in said subject.20. Modified PAM according to any of embodiments 1 to 13 or pharmaceutical compositionaccording to any of embodiments 14 to 17 for effecting an increase in the level of PAM and / or the level of alpha-amidating activity in the blood circulation of a subject, particularly by at least 2-fold, particularly at least 3-fold, particularly at least 5-fold, particularly at least 9-fold, particularly at least 10-fold, particularly wherein said increase lasts for at least 2 hours, more particularly at least 5 hours, more particularly at least 10 hours, more particularly at least 24 hours, more particularly at least 48 hours, more particularly at least 72 hours, more particularly at least 96 hours, more particularly at least 120 hours, more particularly at least 144 hours, more particularly at least 168 hours. In the present invention, the subject to be treated with the modified PAM or the pharmaceutical composition according to the present invention is in particular a subject need of increased PAM levels and / or increased bio-ADM levels and / or increased level of another mature peptide,wherein said other peptide is a peptide occurring in vivo in a precursor form comprising a c-terminal glycine which is transformed to the mature form of the peptide that has a c-terminal amide.21. Modified PAM according to any of embodiments 1 to 13 or pharmaceutical compositionaccording to any of embodiments 14 to 17 for use in the treatment of a disease or disorder selected from the group consisting of cardiovascular, kidney, inflammatory, infectious, and metabolic diseases or disorders, dementia, cancer, and other diseases or disorders connectedwith disturbed peptide homeostasis.22. Modified PAM or pharmaceutical composition for use in the treatment of a disease ordisorder according to embodiment 21, wherein the disease or disorder is sepsis.In particular embodiments of the present invention, said modified PAM or 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”. 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.According to the present invention the applied dosage might be a single bolus injection delivering thedescribed amount of the compounds to be applied or a continuous infusion of the compounds deliveringthe desired amount of compounds over a distinct period of time taking into account the velocity ofinfusion. Further embodiments also relate to the present invention:1. Modified peptidyl-glycine alpha-amidating monooxygenase (PAM) having anincreased in vivo half-life compared to unmodified PAM, wherein the enzymatic activity of modified PAM is maintained compared to unmodified PAM.2. A method for increasing the in vivo half-life of PAM, wherein said method comprisesmodifying said PAM, wherein said in vivo half-life is increased compared to unmodified PAM, and wherein the enzymatic activity of PAM is maintained.3. Modified PAM according to embodiment 1 or method according to embodiment 2,wherein PAM is a protein comprising a polypeptide sequence selected from the group comprising SEQ ID Nos 7 to 18, or having at least 85%, particularly at least 90%, more particularly at least 95%, even more particularly at least 99% sequence homology therewith.4. Modified PAM according to embodiment 1 or method according to embodiment 2,wherein PAM is a protein comprising a polypeptide sequence selected from the group comprising SEQ ID Nos 7 to 16, or having at least 85%, particularly at least 90%, more particularly at least 95%, even more particularly at least 99% sequence homology therewith.5. Modified PAM or method according to any of the preceding embodiments, wherein theenzymatic activity of PAM is the alpha-amidating activity of PAM.6. Modified PAM or method according to any of the preceding embodiments, whereinPAM is modified bya. attaching one or more natural or synthetic polymers units, wherein said polymers areselected from the group comprisingi. PEG, particularly having an average molecular weight in the range of 0.2-100 kDa,particularly 1-90 kDa, more particularly 2-80 kDa, more particularly 3-70 kDa, more particularly 4-60 kDa and even more particularly 5 to 50kDa;ii. XTEN;iii. a peptide polymer consisting of repeats of PAS (the amino acids proline, alanine andserine, particularly comprising 100-200, particularly 120-180, more particularly 130-170, even more particularly about 150 PAS repeats;iv. elastin-like polypeptides consisting of Valin-Prolin-Glycine-x-Glycine repeats, whereinx is any amino acid other than Proline, particularly consisting of from 150, 200, 250, 300, 400, or 500 to 550, 600, 750, 850, 950 or 1,000 amino acids;v. HAP polypeptide, having the sequence (Gly4Ser)n, wherein n is 100-200;vi. gelatin-like fusion protein, having the sequence (Gly-X-Y)n, wherein X and Yindividually are any amino acid except for Cysteine, and wherein n = 60 to 1500;vii. polysaccharides, in particular those chosen from the group comprising:1. dextrans;2. hydroxyethyls;3. heparosan;4. hyaluronic acid;viii. polysialic acid.b. conjugation with a serum protein, such as albumin or an immunoglobulin or parts of animmunoglobulin;c. site-directed mutagenesis by insertion, deletion and / or exchange of one or more aminoacids within the amino acid sequence of PAM.7. Modified PAM or method according to any of the preceding embodiments, wherein thein vivo half-life of said modified PAM, compared with unmodified PAM, is increased by at least 2-fold, particularly at least 5-fold, more particularly at least 10-fold, more particularly at least 20-fold, more particularly at least 50-fold, more particularly at least 100-fold, more particularly at least 150-fold, more particularly at least 250-fold, more particularly at least 350- fold, and / or wherein the in vivo half-life of said modified PAM, compared with unmodified PAM, is increased by at least 0.5 hours, more particularly at least 1 hour, more particularly at least 2 hours, more particularly at least 5 hours, more particularly at least 10 hours, more particularly at least 24 hours, more particularly at least 48 hours, more particularly at least 60 hours and / or wherein the in vivo half-life of said modified PAM is at least 1 hour, more particularly 2 hours, more particularly at least 5 hours, more particularly at least 10 hours, more particularly at least 24 hours, more particularly at least 48 hours, more particularly at least 60 hours.8. Modified PAM or method according to any of the preceding embodiments, wherein thelevel of PAM is increased in a sample obtained from a subject having received at least one dose of said modified PAM, compared to a pre-dose sample obtained from said subject before having received said at least one dose of said modified PAM, wherein said sample is obtained from said subject 2 hours, particularly 5 hours, more particularly 10 hours, more particularly 24 hours, more particularly 48 hours, more particularly 72 hours, more particularly 96 hours, more particularly 120 hours, more particularly 144 hours, more particularly 168 hours after administration of the last dose of said modified PAM to said subject.9. Modified PAM or method according to any of the preceding embodiments, wherein atleast 50%, particularly at least 75%, particularly at least 85%, particularly at least 95%, particularly about 100% of the enzymatic activity is maintained, compared to unmodified PAM.10. Modified PAM or method according to any of the preceding embodiments, wherein saidmodified PAM, when administered to a subject, increases the bioavailability of one or more amidated peptides, in particular one or more peptide hormones in said subject.11. Pharmaceutical composition comprising a modified PAM according to any ofembodiments 1 to 10.12. Pharmaceutical composition according to embodiment 11, which is a ready-to-useformulation.13. Pharmaceutical composition according to any of embodiments 11 to 12, which is afreeze-dried composition.14. Modified PAM according to any of embodiments 1 to 10 or pharmaceutical compositionaccording to any of embodiments 11 to 13 for effecting an increase in the level of PAM and / or the level of alpha-amidating activity in the blood circulation of a subject, particularly by at least 2-fold, particularly at least 3-fold, particularly at least 5-fold, particularly at least 9-fold, particularly at least 10-fold, particularly wherein said increase lasts for at least 2 hours, more particularly at least 5 hours, more particularly at least 10 hours, more particularly at least 24 hours, more particularly at least 48 hours, more particularly at least 72 hours, more particularly at least 96 hours, more particularly at least 120 hours, more particularly at least 144 hours, more particularly at least 168 hours.15. Modified PAM according to any of embodiments 1 to 10 or pharmaceutical compositionaccording to any of embodiments 11 to 13 for use as a medicament, particularly for use in the treatment of a disease or disorder selected from the group consisting of cardiovascular, kidney, inflammatory, infectious, and metabolic diseases or disorders, dementia, cancer, and other diseases or disorders connected with disturbed peptide homeostasis, wherein more particularly said disease or disorder is sepsis.16. Modified PAM according to any of embodiments 1 to 10 or pharmaceutical compositionaccording to any of embodiments 11 to 13 for reducing the risk of a subject for suffering from a medical condition, and / or for reducing the occurrence of a disease or disorder in said subject, and / or for reducing the severity of a disease or disorder in said subject.17. Modified PAM according to any of embodiments 1 to 10 or pharmaceuticalcomposition according to any of embodiments 11 to 13 for use in treatment or prevention of a disease or medical condition in a subject, wherein said disease or medical condition is associated with endothelial barrier dysfunction and / or blood-brain barrier dysfunction.18. Modified PAM for use in treatment or prevention of a disease in a subject according toembodiment 17, wherein said disease or medical condition is selected from the group consistingof cardiovascular, kidney, inflammatory, infectious, and metabolic diseases or disorders, dementia, cancer, and other diseases or medical conditions connected with disturbed peptide homeostasis, wherein more particularly said disease or disorder is sepsis. EXAMPLESExample 1- PAM activity in sepsis and septic shockPAM activity (AMA) was measured in Li-heparin plasma in a randomly chosen cohort of 199individuals of prospective observational multinational Adrenomedullin and Outcome in Sepsisand Septic Shock 1 study (AdrenOSS-1). A cohort comprising 98 self-reported healthyindividuals was used as control group. AMA was determined by the method described inExample 3.The levels of AMA in circulation were measured in Li-Heparin plasma upon admission to theintensive care unit (ICU) in circulation. Median AMA levels in sepsis and septic shock werealso significantly elevated, with levels of 23,5 Units / L [IQD: 17,7 – 32,7 Units / L] and 22,4Units / L [IQD: 18,2 – 38,9 Units / L], respectively, compared to healthy individuals at 18,4Units / L [13,5 – 21,9 Units / L] (Figure 4A). Patients with AMA levels higher than 35.1 Units / Lupon ICU admission had a survival outcome of approximately 15% in sepsis and septic shock,while higher amidating activity led to a mortality rate of ca. 40% (Figure 4B).Example 2 – Determination of ADM-Gly concentration in sepsis and septic shockADM-Gly concentration was measured in Li-heparin plasma in a randomly chosen cohort of199 individuals of the AdrenOSS-1 study. A cohort comprising 100 self-reported healthyindividuals was used as control group. The ADM-Gly concentration was measured in a one-step high throughput microtiter plate-based chemiluminescence immunometric assay with solidphase and tracer antibodies being specifically directed against the mid-regional (amino acids21-42) and C-terminal (amino acids 42-52) part of ADM-Gly, respectively. The levels of ADM-Gly in circulation were measured in Li-Heparin plasma upon admission to the ICU. The median concentration of ADM-Gly was significantly elevated in patients withsepsis (121,5 pg / mL [IQD: 44,4 – 284,1 pg / mL]) and septic shock (419,3 pg / mL [IQD: 148,8– 1106 pg / mL]), when compared to the self-reported healthy individuals (17,5 pg / mL [IQD:11,5 – 27,2 pg / mL]) (Figure 5A). Elevated levels of ADM-Gly were found to be associatedwith higher mortality rates in sepsis and septic shock. Patients with ADM-Gly values lower than 730 pg / mL upon ICU admission had a better survivalprognosis of approximately 15% mortality in both sepsis and septic shock (Figure 5B). Incontrast, the 28-day mortality for the patients with an ADM-Gly value higher than 730 pg / mLwas approx. 50% (Figure 5B).Example 3 – PAM activity assayHuman serum or Li-Heparin plasma from self-reported healthy volunteers was used as sourceof human native PAM. Each sample (20µl) was diluted two-fold in 100 mM Tris-HCl buffersolution in duplicate. The amidation reaction was initiated by addition of 160 µl of PAM-reaction buffer (100 mM Tris-HCl, pH 7.5, 6.25 µM CuSO4, 2.5 mM L-ascorbate, 125 µg / mLcatalase, 62.5 µM amastatin, 250 µM leupeptin, 36 ng / mL synthetic ADM-Gly and 375 µg / mLNT-ADM antibody). Afterwards, 100 µl of each individual reaction of duplicated samples werecombined and transferred into 20 µl of 200 mM EDTA solution to terminate the amidationreaction and to generate t=0 minutes reaction time-point followed by incubation at 37 °C for 40minutes, upon which the non-terminated reactions were stopped by addition of 10µl of 200 mMEDTA solution. To determine the PAM activity, bio-ADM as reaction product was quantifiedin each sample using the sphingotest® bio-ADM immunoassay (Weber et al. 2017). Theamidation 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 thePAM activity (AMA) in the samples. AMA is specified in Units, wherein 1 AMA Unit isdefined as 1 µg bio-ADM formed per hour. Units / L describe AMA as 1 µg bio-ADM formed per hour and L of sample. A typical PAM calibration curve is shown in Figure 6. The distribution of AMAs in Li-Heparin samples from n=120 self-reported healthy volunteers are shown in Figure 7. The median [IQR]of Li-Heparin AMA was 18.4 Units / L [13.5-21.9]. The 10th and 90th percentile were 10.5 and24.2 Units / L, respectively. The 2.5th, 97.5thand 99thpercentile were 8.1, 31.6 and 40.8 Units / L, respectively. In addition, matched serum samples from n=20 subjects were measured andrevealed a highly significant correlation (r = 0.89; p < 0.0001), although AMAs values in serumwere approximately 40% lower when compared to Li-Heparin.Example 4 – Production of recombinant PAMVariant A: PAM cDNA was synthesized according to Uniprot Accession No. P19021 encoding amino acids 21 834 of the PAM protein involving codon optimization for expression in mammalian cells. The signal sequence of PAM was replaced with human serum albumin signal sequence (MKWVTFISLLFLFSSAYSFR [SEQ ID No.9]). At the C-terminus of PAM a hexa- histidine tag was added linked via a GS linker to PAM. The sequence of recombinant PAM (amino acids 21-834 of PAM without signal sequence and hexa-histidine tag) is shown in SEQ ID No. 10. The cDNA was cloned into an expression vector (plasmid DNA) using a 5’-NotI and a 3’ HindIII restriction site. The expression vector harboring the cDNA for PAM expressionwas replicated in- and prepared from E. coli. as a low-endotoxin preparation.HEK-INV cells were transfected with the expression vector using INVect transfection reagents in serum free suspension culture. The transfection rate was controlled via co-transfection witha GFP- (green fluorescent protein) containing expression vector. Cultivation of cells was carriedout in presence of valproic acid and Penicillin-Streptomycin at 37°C and 5% CO2. Cells were harvested via centrifugation when viability reached <60% (>2000g, 30-45 min, 2-8°C). Cell culture supernatant (CCS) was washed 5 times with 100 mM Tris / HCl, pH 8.0 via tangential flow filtration (TFF, 30 kDa cut-off). Purification of recombinant PAM included application of buffer exchanged CCS on a Q- sepharose fast flow resin (GE Healthcare) with a NaCl gradient (up to 2 M) elution. Amidating activity containing fractions were pooled and applied onto a Superdex 200pg (GE Healthcare)size exclusion chromatography column with a 100 mM Tris / HCl, 200 mM NaCl, pH 8.0 elutionbuffer. Amidating activity containing fractions were pooled, dialyzed against 100 mM Tris HCl, 200 mM NaCl, pH 8.0, sterile filtered (0.2 µm). Endotoxin load was determined by Charles River PTS Endosafe system and was below 5 EU / mL. Variant B: The second construct of full-length PAM for immunization was commercially obtained from SinoBiological, comprised 31-818 residues of human PAM (UniProtKB: P19021-1, SEQ ID No.1) and was C-terminally tagged with decahistidine tag. Example 5: Modification of PAM: Pegylation with PEG-5000 Pegylation is a widely used method to improve the pharmacokinetics of peptides and proteins by increasing their half-life. The pegylation protocol described below provides a reproducible and efficient method for pegylation of recombinant PAM using PEG-5000. The protocol: 10 mg of lyophilized PAM was dissolved in 5 mL of phosphate-buffered saline (PBS) at pH 10. Next, 1 M NaOH was added to adjust the pH to 8.5. A 20 mM solution of PEG- 5000 was prepared by dissolving 70.5 mg of PEG-5000 in 705 µL of 20% DMSO. This solution was added to the PAM solution at a molar excess of approximately 90-120-fold compared to PAM, or a 2.2-fold molar excess compared to the lysine residues present in the PAM construct. The PAM-PEG mixture was incubated for 120 minutes at 4°C with constant shaking. After 90 minutes, 500 µL of 1 M Tris solution was added to the mixture to inactivate any unreacted PEG, and incubated for an additional 30 minutes. To separate PEG-PAM from free PEG, the mixture was fractionated on the Superdex200 column (ÄKTA Start) with PBS as the running buffer. The protein peak was detected at 280 nm, and fractions corresponding to a molecular weight of 160 to 90 kDa were collected and pooled. The pooled fractions were sterile filtered with a 0.2 µM filter and aliquoted under sterile conditions for further application in in vivo experiments. Results: the gel filtration analysis revealed a broad peak eluting between 200 kDa and approximately 80 kDa, suggesting varying degrees of pegylation of PAM (Figure 8). This finding was further corroborated by the SDS-PAGE results, which showed a PEG-PAM smear indicative of different degrees of pegylation. The highest UV absorption peak at 280 nm corresponded to a molecular weight of 130 kDa, indicating successful pegylation and an increase in molecular weight. Furthermore, the absence of high-molecular-weight compounds in the UV profile suggests that the current pegylation conditions do not induce protein aggregation. Moreover, the enzyme activity of PEG-PAM was comparable to that of untreated PAM, indicating that the pegylation protocol did not interfere with the enzyme's activity. To this end, the protein concentration of pegylated (PEG-PAM) and non-modified recombinantPAM was determined using the bicinchoninic acid kit (Micro BCA™ Protein-Assay-Kit,ThermoFisher Scientific, Cat# 23235) for protein determination. To evaluate their respectiveactivities, three samples of pegylated PAM and non-modified PAM were prepared, each containing concentrations of 2 µg / mL, 1 µg / mL, and 0.5 µg / mL. The activity of the samples was measured according to the method described in Example 3.The results, as illustrated in Figure 9 revealed that the activity of pegylated PAM was slightlyreduced compared to non-modified PAM. Specifically, when the PAM concentration was 2µg / mL, the pegylated PAM exhibited a decrease in activity by 14.2%. Similarly, at a concentration of 1 µg / mL, the pegylated PAM displayed a reduction in activity by 6.1%. Lastly, for the samples with a concentration of 0.5 µg / mL, the pegylated PAM demonstrated a decrease in activity by 9.3%. Example 6: Single intravenous injection of untreated and pegylated PAMIn vivo half-life time of pegylated PAM (PEG-PAM) and unmodified PAM (Example 4,Variant B) was compared in blood circulation in rat. A single-dose intravenous injection of 14.4Units per kg of animal weight (appr. corresponded to 6 Units per rat) was administered. Li-heparin plasma was collected at 20 min, 40 min, 60 min, 80 min, 120 min, 180 min, 240 min, and 300 min after the i.v. injection, and the PAM amidating activity was measured using the method described in Example 3. Plasma samples were collected from three rats per time point. Results: PEG-PAM exhibited high amidating activity even after 300 min following the bolus injection (Figure 10). In comparison, the unmodified PAM lost 75% of its original activity after 180 mins following the bolus injection. This finding was in agreement with the previously reported half-life time of unmodified PAM in circulation, which was 47 minutes (Kaufmann et al., 2021). Surprisingly, the half-life time of PEG-PAM was found to be greater than 300 min, which corresponded to more than a six-fold increase in half-life time compared to unmodified PAM. Example 7: Administration routes of and half-life time of PEG-PAM in circulation The enrichment of plasmatic amidating activity (AMA) in rats after administration of PEG- PAM via subcutaneous (s.c.), intramuscular (i.m.), and intraperitoneal (i.p.) routes was investigated. A comparative control using untreated PAM (Example 4, Variant B) was alsoincluded. The dosage used was 14.4 Units of PEG-PAM or unmodified PAM per kg of animalweight, corresponding to approximately 6 Units per rat. Li-heparin plasma was withdrawn atvarious time points, including 15 min, 30 min, 60 min, 120 min, 4h, 8 h, 12 h, 24h and 72 hours post bolus injection, and AMA was measured as described in Example 3. Three rats were sampled for plasma per time point. The half-life of modified (pegylated) and unmodified PAM is described as the time required to reduce its activity by half after reaching the maximum plateau following bolus administration (Table 1). Table 1: Half-life time of unmodified and modified PAM (pegylated) in circulation after intravenous, intraperitoneal, subcutaneous and intramuscular bolus administration. Administration route Half-life time (h)i.v. (not modified PAM) 0.5i.v. (modified PAM) 1.63s.c. (modified PAM) 61.5i.p. (modified PAM) 28.6i.m. (modified PAM) 37.9Figure 11 shows a significant increase in plasmatic AMA after 2 hours post s.c. bolus injectionof PEG-PAM, which was 40% above the activity measured prior to bolus injection. After 4 hours, plasmatic AMA was 110% higher compared to the baseline and reached its maximum after 24 hours post-injection with a 984% increase compared to the baseline activity. The AMA remained elevated even after 3 days post-injection, with a 585% higher level compared to pre-injection AMA. In comparison, the unmodified PAM showed a small activity increase of 46%after 4 hours post-bolus injection, returning to basal levels after 8 hours. Similarly, an increase in plasmatic AMA when PEG-PAM was administered i.p. was observed (Figure 12). After 15 minutes post i.p. bolus injection, a 40% increase in AMA was measured in plasma, reaching its highest activity after 8 hours post-bolus injection with a 1900% increase compared to the baseline amidating activity. This AMA increase remained unchanged for the next 16 hours, with AMA still 710% elevated even after 3 days post-injection. The i.p. injection of unmodified PAM showed a small, continuous increase in plasmatic AMA already after 15 minutes of injection, with the highest AMA being reached after 4 hours after the injection, showing a 55% increase compared to baseline activity, returning to basal activity after 8 hours after injection. Additionally, the administration of PEG-PAM via i.m. route also resulted in an increase in plasmatic AMA (Figure 13). A 153% increase in AMA was observed already after 15 minutes post i.m. bolus injection, reaching its highest point of 1680% increases after 4 hours post-bolus injection. This AMA increase remained unchanged for the next 20 hours, with AMA still 935% elevated even after 3 days post-injection. The i.m. injection of unmodified PAM showed anincrease to 185% of plasmatic AMA compared to baseline activity within 2 hours of the i.m.injection. The AMA returned to basal activity already after 12 hours after injection. Taken together, these results suggest that all three administration routes are an excellent choice to increase the PAM amidating activity in plasma. Example 8: Protective effect of PEG-PAM on Blood-Brain Barrier 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. 12-15 week old male C57Bl / 6N mice (Charles River, Germany) were used. All procedures were carried out according to the guidelines from the German Society for Animal Science (Gesellschaft für Versuchstierkunde; GV-SOLAS). 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-gaugeneedle. 2- to 3-mm droplet of faecal material was expelled. The incision was closed using 4-0surgical sutures. Mice were fluid-resuscitated with 500 µl 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 followed- up for 24-hours, comprising n=12 mice per group (1 treatment group, 1 placebo group, 1 treatment-sham group, 1 placebo-sham group). The protective effect of PEGylated PAM on the integrity of the blood-brain barrier was investigated.1xPBS was used in the Placebo groups. The dosing of Placebo or the PEG-PAM was given at day -1 before CLP via the intraperitoneal (i.p.) route of administration. The dosage of the PEG-PAM 14.4 Units / kg of PEG-PAM. 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 1mg / 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 1xPBS. The homogenates were centrifuged for 20 min at 10,000 × g. The supernatants were measured for absorbance at 620 nm to quantify the presence of EB-dye using known EB-dye concentrations as standard.Figure 14 demonstrates that pre-treatment with PEG-PAM in the rodent model withcompromised vascular integrity can prevent the breakdown of the blood-brain barrier induced by systemic inflammation caused by sepsis. The degree of blood-brain barrier leakage, expressed as the concentration of Evans Blue dye in µg / mL per 1 g brain homogenates, was comparable in the SHAM groups treated with PEG-PAM or PBS, as well as in the CLP-induced group treated with PAM (approximately 4 µg EB / g brain homogenate). In contrast, the CLP- induced group treated with PBS exhibited a higher concentration of approximately 13 µg EB / g homogenate. The resulting data clearly indicates that a treatment with PEG-PAM 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. Example 9: Modification of PAM: Pegylation with PEG-10000, PEG-5000 via Cysteines and with an XTEN peptide The modification protocols described below provide a reproducible and efficient method for modification of recombinant PAM (example 4) using Mal-PEG-5000 (5 kDa PEG), NHS-PEG-10000 (10 kDa PEG) and / or an XTEN peptide (SEQ ID No. 19).The protocol: 1.5 mg of recombinant PAM in a total volume of 1.5 mL of phosphate-buffered saline (PBS) at pH 10 was used. 1 M NaOH was added to adjust the pH to 8.5 and the preparation was divided into three equal portions, 500 µl each. For pegylation with NHS-PEG-10000 (11153 Da, Iris Biotech GmbH, Germany), a 10 mM solution of NHS-PEG-10000 was prepared by dissolving 62 mg of PEG-10000 in 556 µL of 20% DMSO. This solution was added to the PAM solution at a molar excess of approximately 90-120-fold compared to PAM, or a 2.2-fold molar excess compared to the lysine residues present in the PAM construct. The PAM-PEG mixture was incubated for 120 minutes at 4°Cwith constant shaking. After 120 minutes, 50 µL of 2 M Tris solution was added to the mixtureto inactivate any unreacted NHS-PEG, and incubated for an additional 30 minutes. To separate PEG-PAM from free PEG, the mixture was fractionated on the Superdex200 column (ÄKTA Start) with PBS as the running buffer. The protein peak was detected at 280 nm, and fractions corresponding to a molecular weight of 160 to 90 kDa were collected and pooled. The pooled fractions were sterile filtered with a 0.2 µM filter and aliquoted under sterile conditions. For pegylation with MAL-PEG-5000 (4908 Da, Iris Biotech GmbH, Germany), a 1M solutionof DTT was prepared and 5µl of the solution were added to the second 500 µl portion of PAM.After an incubation time of 1h at ambient temperature, the PAM protein underwent desalting using a G5 desalting column (EMP Biotech, Germany) resulting in a final volume of 1 mL. A 20 mM solution of MAL-PEG-5000 was prepared by dissolving 65 mg of MAL-PEG in 662,2 µl of 20% DMSO. This solution was added to the PAM solution at a molar excess of approximately 90-120-fold compared to PAM. After an incubation for 120 minutes at ambient temperature under constant shaking and protection from light, PEG-PAM was separated from free PEG by fractionation on the Superdex200 column (ÄKTA Start) with PBS as the running buffer. The protein peak was detected at 280 nm, and fractions corresponding to a molecular weight of 160 to 90 kDa were collected and pooled. The pooled fractions were sterile filtered with a 0.2 µM filter and aliquoted under sterile conditions. For XTENylation with an XTEN peptide having the following amino-acid sequence: H-SESATPESGPGSEPATSGSETPGTSESATPESC-OH (SEQ ID No. 19) (3623,7 Da,custom synthesis by Peptides&Elephants GmbH, Germany), the third 500 µl portion of PAMwas used. In the first step PAM was coupled to the linker MAL-beta-Ala-Osu (Maleimidobutyryloxysuccinimidylester, Iris Biotech GmbH, Germany) by adding of 15 µl of a 10 mM MAL-beta-Ala-Osu (in 20% DMSO) solution to the PAM protein to couple the linker via its N-succinimidylester to free lysine residues of the PAM protein. The reaction was incubated for 120 minutes at 4°C with constant shaking. After 120 minutes, 50 µL of 2 M Tris solution was added to the mixture to inactivate any unreacted linker and incubated for additional 30 minutes. Afterwards the PAM protein underwent desalting into PBS pH 10 using a G5 desalting column (EMP Biotech, Germany) to remove the unreacted linker resulting in a final volume of 1 mL. A 19.3 mM solution of the XTEN-peptide was prepared in PBS pH 10 and a 100-fold molar excess of the XTEN-peptide was added to the PAM-Linker complex to couple the free C-terminal Cysteine residue of the XTEN-peptide to the maleimide moiety of the linker. After an incubation of 120 minutes at ambient temperature under constant shaking and protection from light, XTENylated PAM (X-PAM) was separated from free XTEN by fractionation on the Superdex200 column (ÄKTA Start) with PBS as the running buffer. The protein peak was detected at 280 nm, and fractions corresponding to a molecular weight of 160 to 90 kDa were collected and pooled. The pooled fractions were sterile filtered with a 0.2 µM filter and aliquoted under sterile conditions. The protein concentration of all three PAM preparations was determined using thebicinchoninic acid kit (Micro BCA™ Protein-Assay-Kit, ThermoFisher Scientific, Cat# 23235)for protein determination. Results: the resulting protein preparations (PEG-PAM 5000 (PEGylation via Cysteines), PEG- PAM 10000 (PEGylation via Lysines) and X-PAM (XTENylation via Lysines) were analysed by SDS-PAGE, showing a shift towards a higher molecular weight in all three preparations, when compared to untreated PAM (figure 15). Moreover, the enzyme activity of all preparations was determined as described in example 3 and compared to that of untreated PAM (figure 15). The PEGylation via Lysines using PEG-10000 retains 88% of specific PAM activity when compared to the untreated enzyme. XTENylation via Lysines using a linker retains 76% of specific PAM activity when compared to untreated PAM, indicating that similar toPEGylation with PEG 5000 (example 5) the PEGylation with PEG 10000 does not substantiallyinterfere with PAM activity. XTENylation resulted in a decline of specific PAM activity by 24% when compared to untreated PAM, however, this reduction may be in part caused byoverestimation of total PAM concentration in the used total protein determination assay (MicroBCA™ Protein-Assay-Kit), which may be false-positively impacted by the attached XTENpeptide, leading to an underestimation of specific PAM activity. PEGylation with PEG-5000 via Cysteine residues surprisingly resulted in significant drop of specific PAM activity, indicating that this modification method is not suitable for the enzyme PAM. In summary, the results, as illustrated in figure 15, revealed that the activity of pegylated PAM with PEG 10000 via coupling to free Lysine residues was slightly reduced compared to non- modified PAM. The pegylated PAM exhibited a decrease in activity by 12%. Similarly, coupling of the XTEN peptide to free Lysine residues exhibited a decrease in activity by 24%. Lastly and surprisingly, in contrast to coupling of PEG 5000 to free lysine residues, as shownin example 5, coupling of PEG 5000 to cysteines results in a substantial drop of specific PAMactivity by 87%.SEQ ID NO: 1 - Prepro-PAM isoform 1 AS 1-97310 20 30 40 50 MAGRVPSLLV LLVFPSSCLA FRSPLSVFKR FKETTRPFSN ECLGTTRPVV 60 70 80 90 100 PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR IPVDEEAFVI DFKPRASMDT 110 120 130 140 150 VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA NILYAWARNA PPTRLPKGVG 160 170 180 190 200 FRVGGETGSK YFVLQVHYGD ISAFRDNNKD CSGVSLHLTR LPQPLIAGMY 210 220 230 240 250 LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH VFAYRVHTHH LGKVVSGYRV 260 270 280 290 300 RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF GDLLAARCVF TGEGRTEATH 310 320 330 340 350 IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT QNVAPDMFRT IPPEANIPIP 360 370 380 390 400 VKSDMVMMHE HHKETEYKDK IPLLQQPKRE EEEVLDQGDF YSLLSKLLGE 410 420 430 440 450 REDVVHVHKY NPTEKAESES DLVAEIANVV QKKDLGRSDA REGAEHERGN 460 470 480 490 500 AILVRDRIHK FHRLVSTLRP PESRVFSLQQ PPPGEGTWEP EHTGDFHMEE 510 520 530 540 550 ALDWPGVYLL PGQVSGVALD PKNNLVIFHR GDHVWDGNSF DSKFVYQQIG 560 570 580 590 600 LGPIEEDTIL VIDPNNAAVL QSSGKNLFYL PHGLSIDKDG NYWVTDVALH 610 620 630 640 650 QVFKLDPNNK EGPVLILGRS MQPGSDQNHF CQPTDVAVDP GTGAIYVSDG 660 670 680 690 700 YCNSRIVQFS PSGKFITQWG EESSGSSPLP GQFTVPHSLA LVPLLGQLCV 710 720 730 740 750 ADRENGRIQC FKTDTKEFVR EIKHSSFGRN VFAISYIPGL LFAVNGKPHF 760 770 780 790 800 GDQEPVQGFV MNFSNGEIID IFKPVRKHFD MPHDIVASED GTVYIGDAHT 810 820 830 840 850 NTVWKFTLTE KLEHRSVKKA GIEVQEIKEA EAVVETKMEN KPTSSELQKM 860 870 880 890 900 QEKQKLIKEP GSGVPVVLIT TLLVIPVVVL LAIAIFIRWK KSRAFGDSEH 910 920 930 940 950 KLETSSGRVL GRFRGKGSGG LNLGNFFASR KGYSRKGFDR LSTEGSDQEK 960 970 EDDGSESEEE YSAPLPALAP SSSSEQ ID NO: 2 - Prepro-PAM isoform 2 AS 1-86810 20 30 40 50 MAGRVPSLLV LLVFPSSCLA FRSPLSVFKR FKETTRPFSN ECLGTTRPVV 60 70 80 90 100 PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR IPVDEEAFVI DFKPRASMDT 110 120 130 140 150 VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA NILYAWARNA PPTRLPKGVG 160 170 180 190 200 FRVGGETGSK YFVLQVHYGD ISAFRDNNKD CSGVSLHLTR LPQPLIAGMY 210 220 230 240 250 LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH VFAYRVHTHH LGKVVSGYRV 260 270 280 290 300 RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF GDLLAARCVF TGEGRTEATH 310 320 330 340 350 IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT QNVAPDMFRT IPPEANIPIP 360 370 380 390 400 VKSDMVMMHE HHKETEYKDK IPLLQQPKRE EEEVLDQDFH MEEALDWPGV 410 420 430 440 450 YLLPGQVSGV ALDPKNNLVI FHRGDHVWDG NSFDSKFVYQ QIGLGPIEED 460 470 480 490 500 TILVIDPNNA AVLQSSGKNL FYLPHGLSID KDGNYWVTDV ALHQVFKLDP 510 520 530 540 550 NNKEGPVLIL GRSMQPGSDQ NHFCQPTDVA VDPGTGAIYV SDGYCNSRIV 560 570 580 590 600 QFSPSGKFIT QWGEESSGSS PLPGQFTVPH SLALVPLLGQ LCVADRENGR 610 620 630 640 650 IQCFKTDTKE FVREIKHSSF GRNVFAISYI PGLLFAVNGK PHFGDQEPVQ 660 670 680 690 700 GFVMNFSNGE IIDIFKPVRK HFDMPHDIVA SEDGTVYIGD AHTNTVWKFT 710 720 730 740 750 LTEKLEHRSV KKAGIEVQEI KEAEAVVETK MENKPTSSEL QKMQEKQKLI 760 770 780 790 800 KEPGSGVPVV LITTLLVIPV VVLLAIAIFI RWKKSRAFGD SEHKLETSSG 810 820 830 840 850 RVLGRFRGKG SGGLNLGNFF ASRKGYSRKG FDRLSTEGSD QEKEDDGSES 860 866 EEEYSAPLPA LAPSSSSEQ ID No: 3 - Prepro-PAM isoform 3 AS (amino acids 829-896 of SEQ ID No. 1 missing)10 20 30 40 50 MAGRVPSLLV LLVFPSSCLA FRSPLSVFKR FKETTRPFSN ECLGTTRPVV 60 70 80 90 100 PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR IPVDEEAFVI DFKPRASMDT 110 120 130 140 150 VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA NILYAWARNA PPTRLPKGVG 160 170 180 190 200 FRVGGETGSK YFVLQVHYGD ISAFRDNNKD CSGVSLHLTR LPQPLIAGMY 210 220 230 240 250 LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH VFAYRVHTHH LGKVVSGYRV 260 270 280 290 300 RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF GDLLAARCVF TGEGRTEATH 310 320 330 340 350 IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT QNVAPDMFRT IPPEANIPIP 360 370 380 390 400 VKSDMVMMHE HHKETEYKDK IPLLQQPKRE EEEVLDQGDF YSLLSKLLGE 410 420 430 440 450 REDVVHVHKY NPTEKAESES DLVAEIANVV QKKDLGRSDA REGAEHERGN 460 470 480 490 500 AILVRDRIHK FHRLVSTLRP PESRVFSLQQ PPPGEGTWEP EHTGDFHMEE 510 520 530 540 550 ALDWPGVYLL PGQVSGVALD PKNNLVIFHR GDHVWDGNSF DSKFVYQQIG 560 570 580 590 600 LGPIEEDTIL VIDPNNAAVL QSSGKNLFYL PHGLSIDKDG NYWVTDVALH 610 620 630 640 650 QVFKLDPNNK EGPVLILGRS MQPGSDQNHF CQPTDVAVDP GTGAIYVSDG 660 670 680 690 700 YCNSRIVQFS PSGKFITQWG EESSGSSPLP GQFTVPHSLA LVPLLGQLCV 710 720 730 740 750 ADRENGRIQC FKTDTKEFVR EIKHSSFGRN VFAISYIPGL LFAVNGKPHF 760 770 780 790 800 GDQEPVQGFV MNFSNGEIID IFKPVRKHFD MPHDIVASED GTVYIGDAHT 810 820 830 840 850 NTVWKFTLTE KLEHRSVKKA GIEVQEIKDS EHKLETSSGR VLGRFRGKGS 860 870 880 890 900 GGLNLGNFFA SRKGYSRKGF DRLSTEGSDQ EKEDDGSESE EEYSAPLPAL 905 APSSSSEQ ID No: 4 - Prepro-PAM isoform 4 (amino acids 829-914 of SEQ ID No. 1 missing)10 20 30 40 50 MAGRVPSLLV LLVFPSSCLA FRSPLSVFKR FKETTRPFSN ECLGTTRPVV 60 70 80 90 100 PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR IPVDEEAFVI DFKPRASMDT 110 120 130 140 150 VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA NILYAWARNA PPTRLPKGVG 160 170 180 190 200 FRVGGETGSK YFVLQVHYGD ISAFRDNNKD CSGVSLHLTR LPQPLIAGMY 210 220 230 240 250 LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH VFAYRVHTHH LGKVVSGYRV 260 270 280 290 300 RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF GDLLAARCVF TGEGRTEATH 310 320 330 340 350 IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT QNVAPDMFRT IPPEANIPIP 360 370 380 390 400 VKSDMVMMHE HHKETEYKDK IPLLQQPKRE EEEVLDQGDF YSLLSKLLGE 410 420 430 440 450 REDVVHVHKY NPTEKAESES DLVAEIANVV QKKDLGRSDA REGAEHERGN 460 470 480 490 500 AILVRDRIHK FHRLVSTLRP PESRVFSLQQ PPPGEGTWEP EHTGDFHMEE 510 520 530 540 550 ALDWPGVYLL PGQVSGVALD PKNNLVIFHR GDHVWDGNSF DSKFVYQQIG 560 570 580 590 600 LGPIEEDTIL VIDPNNAAVL QSSGKNLFYL PHGLSIDKDG NYWVTDVALH 610 620 630 640 650 QVFKLDPNNK EGPVLILGRS MQPGSDQNHF CQPTDVAVDP GTGAIYVSDG 660 670 680 690 700 YCNSRIVQFS PSGKFITQWG EESSGSSPLP GQFTVPHSLA LVPLLGQLCV 710 720 730 740 750 ADRENGRIQC FKTDTKEFVR EIKHSSFGRN VFAISYIPGL LFAVNGKPHF 760 770 780 790 800 GDQEPVQGFV MNFSNGEIID IFKPVRKHFD MPHDIVASED GTVYIGDAHT 810 820 830 840 850 NTVWKFTLTE KLEHRSVKKA GIEVQEIKGK GSGGLNLGNF FASRKGYSRK 860 870 880 887 GFDRLSTEGS DQEKEDDGSE SEEEYSAPLP ALAPSSSSEQ ID No: 5 - Prepro-PAM Isoform 5 (Isoform 1 with an additional aa in position 896)10 20 30 40 50 MAGRVPSLLV LLVFPSSCLA FRSPLSVFKR FKETTRPFSN ECLGTTRPVV 60 70 80 90 100 PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR IPVDEEAFVI DFKPRASMDT 110 120 130 140 150 VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA NILYAWARNA PPTRLPKGVG 160 170 180 190 200 FRVGGETGSK YFVLQVHYGD ISAFRDNNKD CSGVSLHLTR LPQPLIAGMY 210 220 230 240 250 LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH VFAYRVHTHH LGKVVSGYRV 260 270 280 290 300 RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF GDLLAARCVF TGEGRTEATH 310 320 330 340 350 IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT QNVAPDMFRT IPPEANIPIP 360 370 380 390 400 VKSDMVMMHE HHKETEYKDK IPLLQQPKRE EEEVLDQGDF YSLLSKLLGE 410 420 430 440 450 REDVVHVHKY NPTEKAESES DLVAEIANVV QKKDLGRSDA REGAEHERGN 460 470 480 490 500 AILVRDRIHK FHRLVSTLRP PESRVFSLQQ PPPGEGTWEP EHTGDFHMEE 510 520 530 540 550 ALDWPGVYLL PGQVSGVALD PKNNLVIFHR GDHVWDGNSF DSKFVYQQIG 560 570 580 590 600 LGPIEEDTIL VIDPNNAAVL QSSGKNLFYL PHGLSIDKDG NYWVTDVALH 610 620 630 640 650 QVFKLDPNNK EGPVLILGRS MQPGSDQNHF CQPTDVAVDP GTGAIYVSDG 660 670 680 690 700 YCNSRIVQFS PSGKFITQWG EESSGSSPLP GQFTVPHSLA LVPLLGQLCV 710 720 730 740 750 ADRENGRIQC FKTDTKEFVR EIKHSSFGRN VFAISYIPGL LFAVNGKPHF 760 770 780 790 800 GDQEPVQGFV MNFSNGEIID IFKPVRKHFD MPHDIVASED GTVYIGDAHT 810 820 830 840 850 NTVWKFTLTE KLEHRSVKKA GIEVQEIKEA EAVVETKMEN KPTSSELQKM 860 870 880 890 900 QEKQKLIKEP GSGVPVVLIT TLLVIPVVVL LAIAIFIRWK KSRAFGADSE 910 920 930 940 950 HKLETSSGRV LGRFRGKGSG GLNLGNFFAS RKGYSRKGFD RLSTEGSDQE 960 970 KEDDGSESEE EYSAPLPALA PSSSSEQ ID No: 6 - Prepro-PAM Isoform 6 (amino acids 897-914 of SEQ ID No. 1 missing)10 20 30 40 50 MAGRVPSLLV LLVFPSSCLA FRSPLSVFKR FKETTRPFSN ECLGTTRPVV 60 70 80 90 100 PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR IPVDEEAFVI DFKPRASMDT 110 120 130 140 150 VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA NILYAWARNA PPTRLPKGVG 160 170 180 190 200 FRVGGETGSK YFVLQVHYGD ISAFRDNNKD CSGVSLHLTR LPQPLIAGMY 210 220 230 240 250 LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH VFAYRVHTHH LGKVVSGYRV 260 270 280 290 300 RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF GDLLAARCVF TGEGRTEATH 310 320 330 340 350 IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT QNVAPDMFRT IPPEANIPIP 360 370 380 390 400 VKSDMVMMHE HHKETEYKDK IPLLQQPKRE EEEVLDQGDF YSLLSKLLGE 410 420 430 440 450 REDVVHVHKY NPTEKAESES DLVAEIANVV QKKDLGRSDA REGAEHERGN 460 470 480 490 500 AILVRDRIHK FHRLVSTLRP PESRVFSLQQ PPPGEGTWEP EHTGDFHMEE 510 520 530 540 550 ALDWPGVYLL PGQVSGVALD PKNNLVIFHR GDHVWDGNSF DSKFVYQQIG 560 570 580 590 600 LGPIEEDTIL VIDPNNAAVL QSSGKNLFYL PHGLSIDKDG NYWVTDVALH 610 620 630 640 650 QVFKLDPNNK EGPVLILGRS MQPGSDQNHF CQPTDVAVDP GTGAIYVSDG 660 670 680 690 700 YCNSRIVQFS PSGKFITQWG EESSGSSPLP GQFTVPHSLA LVPLLGQLCV 710 720 730 740 750 ADRENGRIQC FKTDTKEFVR EIKHSSFGRN VFAISYIPGL LFAVNGKPHF 760 770 780 790 800 GDQEPVQGFV MNFSNGEIID IFKPVRKHFD MPHDIVASED GTVYIGDAHT 810 820 830 840 850 NTVWKFTLTE KLEHRSVKKA GIEVQEIKEA EAVVETKMEN KPTSSELQKM 860 870 880 890 900 QEKQKLIKEP GSGVPVVLIT TLLVIPVVVL LAIAIFIRWK KSRAFGGKGS 910 920 930 940 950 GGLNLGNFFA SRKGYSRKGF DRLSTEGSDQ EKEDDGSESE EEYSAPLPAL 955 APSSSSEQ ID No: 7 - PHM subunit of PAM10 20 30 40 50 FKETTRPFSN ECLGTTRPVV PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR 60 70 80 90 100 IPVDEEAFVI DFKPRASMDT VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA 110 120 130 140 150 NILYAWARNA PPTRLPKGVG FRVGGETGSK YFVLQVHYGD ISAFRDNNKD 160 170 180 190 200 CSGVSLHLTR LPQPLIAGMY LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH 210 220 230 240 250 VFAYRVHTHH LGKVVSGYRV RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF 260 270 280 290 300 GDLLAARCVF TGEGRTEATH IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT 310 320 330 340 350 QNVAPDMFRT IPPEANIPIP VKSDMVMMHE HHKETEYKDK IPLLQQPKRE 360 370 380 390 400 EEEVLDQGDF YSLLSKLLGE REDVVHVHKY NPTEKAESES DLVAEIANVV 410 420 430 440 450 QKKDLGRSDA REGAEHERGN AILVRDRIHK FHRLVSTLRP PESRVFSLQQ 460 PPPGEGTWEP EHTGSEQ ID No: 8 - PAL subunit of PAM10 20 30 40 50 DFHMEEALDW PGVYLLPGQV SGVALDPKNN LVIFHRGDHV WDGNSFDSKF 60 70 80 90 100 VYQQIGLGPI EEDTILVIDP NNAAVLQSSG KNLFYLPHGL SIDKDGNYWV 110 120 130 140 150 TDVALHQVFK LDPNNKEGPV LILGRSMQPG SDQNHFCQPT DVAVDPGTGA 160 170 180 190 200 IYVSDGYCNS RIVQFSPSGK FITQWGEESS GSSPLPGQFT VPHSLALVPL 210 220 230 240 250 LGQLCVADRE NGRIQCFKTD TKEFVREIKH SSFGRNVFAI SYIPGLLFAV 260 270 280 290 300 NGKPHFGDQE PVQGFVMNFS NGEIIDIFKP VRKHFDMPHD IVASEDGTVY 310 320IGDAHTNTVW KFTLTEKLEH RSVSEQ ID No. 9 – PHM fragment (aa 31-377 of PAM SEQ ID No. 1)10 20 30 40 50FKETTRPFSN ECLGTTRPVV PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR60 70 80 90 100 IPVDEEAFVI DFKPRASMDT VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA 110 120 130 140 150 NILYAWARNA PPTRLPKGVG FRVGGETGSK YFVLQVHYGD ISAFRDNNKD 160 170 180 190 200 CSGVSLHLTR LPQPLIAGMY LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH 210 220 230 240 250 VFAYRVHTHH LGKVVSGYRV RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF 260 270 280 290 300 GDLLAARCVF TGEGRTEATH IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT 310 320 330 340 347 QNVAPDMFRT IPPEANIPIP VKSDMVMMHE HHKETEYKDK IPLLQQPSEQ ID No: 10 - Sequence of recombinant human PAM fragment (amino acid 23 – 834 ofPAM isoform 1 SEQ ID No.1) with additional C-terminal amino acids GS 10 20 30 40 50 SPLSVFKRFK ETTRPFSNEC LGTTRPVVPI DSSDFALDIR MPGVTPKQSD 60 70 80 90 100 TYFCMSMRIP VDEEAFVIDF KPRASMDTVH HMLLFGCNMP SSTGSYWFCD 110 120 130 140 150 EGTCTDKANI LYAWARNAPP TRLPKGVGFR VGGETGSKYF VLQVHYGDIS 160 170 180 190 200 AFRDNNKDCS GVSLHLTRLP QPLIAGMYLM MSVDTVIPAG EKVVNSDISC 210 220 230 240 250 HYKNYPMHVF AYRVHTHHLG KVVSGYRVRN GQWTLIGRQS PQLPQAFYPV 260 270 280 290 300 GHPVDVSFGD LLAARCVFTG EGRTEATHIG GTSSDEMCNL YIMYYMEAKH 310 320 330 340 350 AVSFMTCTQN VAPDMFRTIP PEANIPIPVK SDMVMMHEHH KETEYKDKIP 360 370 380 390 400 LLQQPKREEE EVLDQGDFYS LLSKLLGERE DVVHVHKYNP TEKAESESDL 410 420 430 440 450 VAEIANVVQK KDLGRSDARE GAEHERGNAI LVRDRIHKFH RLVSTLRPPE 460 470 480 490 500 SRVFSLQQPP PGEGTWEPEH TGDFHMEEAL DWPGVYLLPG QVSGVALDPK 510 520 530 540 550 NNLVIFHRGD HVWDGNSFDS KFVYQQIGLG PIEEDTILVI DPNNAAVLQS 560 570 580 590 600 SGKNLFYLPH GLSIDKDGNY WVTDVALHQV FKLDPNNKEG PVLILGRSMQ 610 620 630 640 650 PGSDQNHFCQ PTDVAVDPGT GAIYVSDGYC NSRIVQFSPS GKFITQWGEE 660 670 680 690 700 SSGSSPLPGQ FTVPHSLALV PLLGQLCVAD RENGRIQCFK TDTKEFVREI 710 720 730 740 750 KHSSFGRNVF AISYIPGLLF AVNGKPHFGD QEPVQGFVMN FSNGEIIDIF 760 770 780 790 800 KPVRKHFDMP HDIVASEDGT VYIGDAHTNT VWKFTLTEKL EHRSVKKAGI 810 EVQEIKEAEA VVGSSEQ ID NO: 11 - PAM isoform 1 AS 31-973 of Prepro-PAM Isoform 1 (amino acids 1-30 ofSEQ ID No.1 missing) 10 20 30 40 50 FKETTRPFSN ECLGTTRPVV PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR 60 70 80 90 100 IPVDEEAFVI DFKPRASMDT VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA 110 120 130 140 150 NILYAWARNA PPTRLPKGVG FRVGGETGSK YFVLQVHYGD ISAFRDNNKD 160 170 180 190 200 CSGVSLHLTR LPQPLIAGMY LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH 210 220 230 240 250 VFAYRVHTHH LGKVVSGYRV RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF 260 270 280 290 300 GDLLAARCVF TGEGRTEATH IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT 310 320 330 340 350 QNVAPDMFRT IPPEANIPIP VKSDMVMMHE HHKETEYKDK IPLLQQPKRE 360 370 380 390 400 EEEVLDQGDF YSLLSKLLGE REDVVHVHKY NPTEKAESES DLVAEIANVV 410 420 430 440 450 QKKDLGRSDA REGAEHERGN AILVRDRIHK FHRLVSTLRP PESRVFSLQQ 460 470 480 490 500 PPPGEGTWEP EHTGDFHMEE ALDWPGVYLL PGQVSGVALD PKNNLVIFHR 510 520 530 540 550 GDHVWDGNSF DSKFVYQQIG LGPIEEDTIL VIDPNNAAVL QSSGKNLFYL 560 570 580 590 600 PHGLSIDKDG NYWVTDVALH QVFKLDPNNK EGPVLILGRS MQPGSDQNHF 610 620 630 640 650 CQPTDVAVDP GTGAIYVSDG YCNSRIVQFS PSGKFITQWG EESSGSSPLP 660 670 680 690 700 GQFTVPHSLA LVPLLGQLCV ADRENGRIQC FKTDTKEFVR EIKHSSFGRN 710 720 730 740 750 VFAISYIPGL LFAVNGKPHF GDQEPVQGFV MNFSNGEIID IFKPVRKHFD 760 770 780 790 800 MPHDIVASED GTVYIGDAHT NTVWKFTLTE KLEHRSVKKA GIEVQEIKEA 810 820 830 840 850 EAVVETKMEN KPTSSELQKM QEKQKLIKEP GSGVPVVLIT TLLVIPVVVL 860 870 880 890 900 LAIAIFIRWK KSRAFGDSEH KLETSSGRVL GRFRGKGSGG LNLGNFFASR 910 920 930 940 943 KGYSRKGFDR LSTEGSDQEK EDDGSESEEE YSAPLPALAP SSSSEQ ID NO: 12 - PAM isoform 2 AS 31-868 of prepro-PAM isoform 2 (amino acids 1-30 ofSEQ ID No.2 missing) 10 20 30 40 50 FKETTRPFSN ECLGTTRPVV PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR 60 70 80 90 100 IPVDEEAFVI DFKPRASMDT VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA 110 120 130 140 150 NILYAWARNA PPTRLPKGVG FRVGGETGSK YFVLQVHYGD ISAFRDNNKD 160 170 180 190 200 CSGVSLHLTR LPQPLIAGMY LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH 210 220 230 240 250 VFAYRVHTHH LGKVVSGYRV RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF 260 270 280 290 300 GDLLAARCVF TGEGRTEATH IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT 310 320 330 340 350 QNVAPDMFRT IPPEANIPIP VKSDMVMMHE HHKETEYKDK IPLLQQPKRE 360 370 380 390 400 EEEVLDQDFH MEEALDWPGV YLLPGQVSGV ALDPKNNLVI FHRGDHVWDG 410 420 430 440 450 NSFDSKFVYQ QIGLGPIEED TILVIDPNNA AVLQSSGKNL FYLPHGLSID 460 470 480 490 500 KDGNYWVTDV ALHQVFKLDP NNKEGPVLIL GRSMQPGSDQ NHFCQPTDVA 510 520 530 540 550 VDPGTGAIYV SDGYCNSRIV QFSPSGKFIT QWGEESSGSS PLPGQFTVPH 560 570 580 590 600 SLALVPLLGQ LCVADRENGR IQCFKTDTKE FVREIKHSSF GRNVFAISYI 610 620 630 640 650 PGLLFAVNGK PHFGDQEPVQ GFVMNFSNGE IIDIFKPVRK HFDMPHDIVA 660 670 680 690 700 SEDGTVYIGD AHTNTVWKFT LTEKLEHRSV KKAGIEVQEI KEAEAVVETK 710 720 730 740 750 MENKPTSSEL QKMQEKQKLI KEPGSGVPVV LITTLLVIPV VVLLAIAIFI 760 770 780 790 800 RWKKSRAFGD SEHKLETSSG RVLGRFRGKG SGGLNLGNFF ASRKGYSRKG 810 820 830 836 FDRLSTEGSD QEKEDDGSES EEEYSAPLPA LAPSSSSEQ ID No: 13 - PAM isoform 3 AS 31-905 of prepro-PAM isoform 3 (amino acids 1-30 ofSEQ ID No. 3 missing)10 20 30 40 50 FKETTRPFSN ECLGTTRPVV PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR 60 70 80 90 100 IPVDEEAFVI DFKPRASMDT VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA 110 120 130 140 150 NILYAWARNA PPTRLPKGVG FRVGGETGSK YFVLQVHYGD ISAFRDNNKD 160 170 180 190 200 CSGVSLHLTR LPQPLIAGMY LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH 210 220 230 240 250 VFAYRVHTHH LGKVVSGYRV RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF 260 270 280 290 300 GDLLAARCVF TGEGRTEATH IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT 310 320 330 340 350 QNVAPDMFRT IPPEANIPIP VKSDMVMMHE HHKETEYKDK IPLLQQPKRE 360 370 380 390 400 EEEVLDQGDF YSLLSKLLGE REDVVHVHKY NPTEKAESES DLVAEIANVV 410 420 430 440 450 QKKDLGRSDA REGAEHERGN AILVRDRIHK FHRLVSTLRP PESRVFSLQQ 460 470 480 490 500 PPPGEGTWEP EHTGDFHMEE ALDWPGVYLL PGQVSGVALD PKNNLVIFHR 510 520 530 540 550 GDHVWDGNSF DSKFVYQQIG LGPIEEDTIL VIDPNNAAVL QSSGKNLFYL 560 570 580 590 600 PHGLSIDKDG NYWVTDVALH QVFKLDPNNK EGPVLILGRS MQPGSDQNHF 610 620 630 640 650 CQPTDVAVDP GTGAIYVSDG YCNSRIVQFS PSGKFITQWG EESSGSSPLP 660 670 680 690 700 GQFTVPHSLA LVPLLGQLCV ADRENGRIQC FKTDTKEFVR EIKHSSFGRN 710 720 730 740 750 VFAISYIPGL LFAVNGKPHF GDQEPVQGFV MNFSNGEIID IFKPVRKHFD 760 770 780 790 800 MPHDIVASED GTVYIGDAHT NTVWKFTLTE KLEHRSVKKA GIEVQEIKDS 810 820 830 840 850 EHKLETSSGR VLGRFRGKGS GGLNLGNFFA SRKGYSRKGF DRLSTEGSDQ 860 870 885 EKEDDGSESE EEYSAPLPAL APSSSSEQ ID No: 14 - PAM isoform 4 AS 31-887 of prepro-PAM isoform 4 (amino acids 1-30 ofSEQ ID No. 4 missing)10 20 30 40 50 FKETTRPFSN ECLGTTRPVV PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR 60 70 80 90 100 IPVDEEAFVI DFKPRASMDT VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA 110 120 130 140 150 NILYAWARNA PPTRLPKGVG FRVGGETGSK YFVLQVHYGD ISAFRDNNKD 160 170 180 190 200 CSGVSLHLTR LPQPLIAGMY LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH 210 220 230 240 250 VFAYRVHTHH LGKVVSGYRV RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF 260 270 280 290 300 GDLLAARCVF TGEGRTEATH IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT 310 320 330 340 350 QNVAPDMFRT IPPEANIPIP VKSDMVMMHE HHKETEYKDK IPLLQQPKRE 360 370 380 390 400 EEEVLDQGDF YSLLSKLLGE REDVVHVHKY NPTEKAESES DLVAEIANVV 410 420 430 440 450 QKKDLGRSDA REGAEHERGN AILVRDRIHK FHRLVSTLRP PESRVFSLQQ 460 470 480 490 500 PPPGEGTWEP EHTGDFHMEE ALDWPGVYLL PGQVSGVALD PKNNLVIFHR 510 520 530 540 550 GDHVWDGNSF DSKFVYQQIG LGPIEEDTIL VIDPNNAAVL QSSGKNLFYL 560 570 580 590 600 PHGLSIDKDG NYWVTDVALH QVFKLDPNNK EGPVLILGRS MQPGSDQNHF 610 620 630 640 650 CQPTDVAVDP GTGAIYVSDG YCNSRIVQFS PSGKFITQWG EESSGSSPLP 660 670 680 690 700 GQFTVPHSLA LVPLLGQLCV ADRENGRIQC FKTDTKEFVR EIKHSSFGRN 710 720 730 740 750 VFAISYIPGL LFAVNGKPHF GDQEPVQGFV MNFSNGEIID IFKPVRKHFD 760 770 780 790 800 MPHDIVASED GTVYIGDAHT NTVWKFTLTE KLEHRSVKKA GIEVQEIKGK 810 820 830 840 850 GSGGLNLGNF FASRKGYSRK GFDRLSTEGS DQEKEDDGSE SEEEYSAPLP 857 ALAPSSSSEQ ID No: 15 - PAM Isoform 5 AS 31-973 of prepro-PAM Isoform 5 (SEQ ID No. 5 withamino acids 1-30 missing) 10 20 30 40 50 FKETTRPFSN ECLGTTRPVV PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR 60 70 80 90 100 IPVDEEAFVI DFKPRASMDT VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA 110 120 130 140 150 NILYAWARNA PPTRLPKGVG FRVGGETGSK YFVLQVHYGD ISAFRDNNKD 160 170 180 190 200 CSGVSLHLTR LPQPLIAGMY LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH 210 220 230 240 250 VFAYRVHTHH LGKVVSGYRV RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF 260 270 280 290 300 GDLLAARCVF TGEGRTEATH IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT 310 320 330 340 350 QNVAPDMFRT IPPEANIPIP VKSDMVMMHE HHKETEYKDK IPLLQQPKRE 360 370 380 390 400 EEEVLDQGDF YSLLSKLLGE REDVVHVHKY NPTEKAESES DLVAEIANVV 410 420 430 440 450 QKKDLGRSDA REGAEHERGN AILVRDRIHK FHRLVSTLRP PESRVFSLQQ 460 470 480 490 500 PPPGEGTWEP EHTGDFHMEE ALDWPGVYLL PGQVSGVALD PKNNLVIFHR 510 520 530 540 550 GDHVWDGNSF DSKFVYQQIG LGPIEEDTIL VIDPNNAAVL QSSGKNLFYL 560 570 580 590 600 PHGLSIDKDG NYWVTDVALH QVFKLDPNNK EGPVLILGRS MQPGSDQNHF 610 620 630 640 650 CQPTDVAVDP GTGAIYVSDG YCNSRIVQFS PSGKFITQWG EESSGSSPLP 660 670 680 690 700 GQFTVPHSLA LVPLLGQLCV ADRENGRIQC FKTDTKEFVR EIKHSSFGRN 710 720 730 740 750 VFAISYIPGL LFAVNGKPHF GDQEPVQGFV MNFSNGEIID IFKPVRKHFD 760 770 780 790 800 MPHDIVASED GTVYIGDAHT NTVWKFTLTE KLEHRSVKKA GIEVQEIKEA 810 820 830 840 850 EAVVETKMEN KPTSSELQKM QEKQKLIKEP GSGVPVVLIT TLLVIPVVVL 860 870 880 890 900 LAIAIFIRWK KSRAFGADSE HKLETSSGRV LGRFRGKGSG GLNLGNFFAS 910 920 930 940 944 RKGYSRKGFD RLSTEGSDQE KEDDGSESEE EYSAPLPALA PSSSSEQ ID No: 16 - PAM Isoform 6 AS 31-955 of prepro-PAM Isoform 6 (amino acids 1-30 ofSEQ ID No. 6 missing)10 20 30 40 50 FKETTRPFSN ECLGTTRPVV PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR 60 70 80 90 100 IPVDEEAFVI DFKPRASMDT VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA 110 120 130 140 150 NILYAWARNA PPTRLPKGVG FRVGGETGSK YFVLQVHYGD ISAFRDNNKD 160 170 180 190 200 CSGVSLHLTR LPQPLIAGMY LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH 210 220 230 240 250 VFAYRVHTHH LGKVVSGYRV RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF 260 270 280 290 300 GDLLAARCVF TGEGRTEATH IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT 310 320 330 340 350 QNVAPDMFRT IPPEANIPIP VKSDMVMMHE HHKETEYKDK IPLLQQPKRE 360 370 380 390 400 EEEVLDQGDF YSLLSKLLGE REDVVHVHKY NPTEKAESES DLVAEIANVV 410 420 430 440 450 QKKDLGRSDA REGAEHERGN AILVRDRIHK FHRLVSTLRP PESRVFSLQQ 460 470 480 490 500 PPPGEGTWEP EHTGDFHMEE ALDWPGVYLL PGQVSGVALD PKNNLVIFHR 510 520 530 540 550 GDHVWDGNSF DSKFVYQQIG LGPIEEDTIL VIDPNNAAVL QSSGKNLFYL 560 570 580 590 600 PHGLSIDKDG NYWVTDVALH QVFKLDPNNK EGPVLILGRS MQPGSDQNHF 610 620 630 640 650 CQPTDVAVDP GTGAIYVSDG YCNSRIVQFS PSGKFITQWG EESSGSSPLP 660 670 680 690 700 GQFTVPHSLA LVPLLGQLCV ADRENGRIQC FKTDTKEFVR EIKHSSFGRN 710 720 730 740 750 VFAISYIPGL LFAVNGKPHF GDQEPVQGFV MNFSNGEIID IFKPVRKHFD 760 770 780 790 800 MPHDIVASED GTVYIGDAHT NTVWKFTLTE KLEHRSVKKA GIEVQEIKEA 810 820 830 840 850 EAVVETKMEN KPTSSELQKM QEKQKLIKEP GSGVPVVLIT TLLVIPVVVL 860 870 880 890 900 LAIAIFIRWK KSRAFGGKGS GGLNLGNFFA SRKGYSRKGF DRLSTEGSDQ 910 920 925 EKEDDGSESE EEYSAPLPAL APSSSSEQ ID No: 17 – sequence of recombinant PAM variant 2 (amino acids 31-387 and 495-817of SEQ ID No.1) 10 20 30 40 50 FKETTRPFSN ECLGTTRPVV PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR 60 70 80 90 100 IPVDEEAFVI DFKPRASMDT VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA 110 120 130 140 150 NILYAWARNA PPTRLPKGVG FRVGGETGSK YFVLQVHYGD ISAFRDNNKD 160 170 180 190 200 CSGVSLHLTR LPQPLIAGMY LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH 210 220 230 240 250 VFAYRVHTHH LGKVVSGYRV RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF 260 270 280 290 300 GDLLAARCVF TGEGRTEATH IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT 310 320 330 340 350 QNVAPDMFRT IPPEANIPIP VKSDMVMMHE HHKETEYKDK IPLLQQPKRE 360 370 380 390 400 EEEVLDQDFH MEEALDWPGV YLLPGQVSGV ALDPKNNLVI FHRGDHVWDG 410 420 430 440 450 NSFDSKFVYQ QIGLGPIEED TILVIDPNNA AVLQSSGKNL FYLPHGLSID 460 470 480 490 500 KDGNYWVTDV ALHQVFKLDP NNKEGPVLIL GRSMQPGSDQ NHFCQPTDVA 510 520 530 540 550 VDPGTGAIYV SDGYCNSRIV QFSPSGKFIT QWGEESSGSS PLPGQFTVPH 560 570 580 590 600 SLALVPLLGQ LCVADRENGR IQCFKTDTKE FVREIKHSSF GRNVFAISYI 610 620 630 640 650 PGLLFAVNGK PHFGDQEPVQ GFVMNFSNGE IIDIFKPVRK HFDMPHDIVA 660 670 680 SEDGTVYIGD AHTNTVWKFT LTEKLEHRSVSEQ ID No. 18 - recombinant PAM AS 31-834 of prepro-PAM Isoform 1 with additional C-terminalamino acids GS 10 20 30 40 50 FKETTRPFSN ECLGTTRPVV PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR 60 70 80 90 100 IPVDEEAFVI DFKPRASMDT VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA 110 120 130 140 150 NILYAWARNA PPTRLPKGVG FRVGGETGSK YFVLQVHYGD ISAFRDNNKD 160 170 180 190 200 CSGVSLHLTR LPQPLIAGMY LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH 210 220 230 240 250 VFAYRVHTHH LGKVVSGYRV RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF 260 270 280 290 300 GDLLAARCVF TGEGRTEATH IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT 310 320 330 340 350 QNVAPDMFRT IPPEANIPIP VKSDMVMMHE HHKETEYKDK IPLLQQPKRE 360 370 380 390 400 EEEVLDQGDF YSLLSKLLGE REDVVHVHKY NPTEKAESES DLVAEIANVV 410 420 430 440 450 QKKDLGRSDA REGAEHERGN AILVRDRIHK FHRLVSTLRP PESRVFSLQQ 460 470 480 490 500 PPPGEGTWEP EHTGDFHMEE ALDWPGVYLL PGQVSGVALD PKNNLVIFHR 510 520 530 540 550 GDHVWDGNSF DSKFVYQQIG LGPIEEDTIL VIDPNNAAVL QSSGKNLFYL 560 570 580 590 600 PHGLSIDKDG NYWVTDVALH QVFKLDPNNK EGPVLILGRS MQPGSDQNHF 610 620 630 640 650 CQPTDVAVDP GTGAIYVSDG YCNSRIVQFS PSGKFITQWG EESSGSSPLP 660 670 680 690 700 GQFTVPHSLA LVPLLGQLCV ADRENGRIQC FKTDTKEFVR EIKHSSFGRN 710 720 730 740 750 VFAISYIPGL LFAVNGKPHF GDQEPVQGFV MNFSNGEIID IFKPVRKHFD 760 770 780 790 800 MPHDIVASED GTVYIGDAHT NTVWKFTLTE KLEHRSVKKA GIEVQEIKEA EAVVGSSEQ ID No. 19 – XTEN peptide10 20 30 40 50SESATPESGP GSEPATSGSE TPGTSESATP ESC

Claims

CLAIMS1. Modified peptidyl-glycine alpha-amidating monooxygenase (PAM) having an increased invivo half-life compared to unmodified PAM, wherein the enzymatic activity of modifiedPAM is maintained compared to unmodified PAM.

2. A method for increasing the in vivo half-life of PAM, wherein said method comprisesmodifying said PAM, wherein said in vivo half-life is increased compared to unmodifiedPAM, and wherein the enzymatic activity of PAM is maintained.

3. Modified PAM according to claim 1 or method according to claim 2, wherein PAM is aprotein comprising a polypeptide sequence selected from the group comprising SEQ ID Nos7 to 18, or having at least 85%, particularly at least 90%, more particularly at least 95%,even more particularly at least 99% sequence homology therewith.

4. Modified PAM according to claim 1 or method according to claim 2, wherein PAM is aprotein comprising a polypeptide sequence selected from the group comprising SEQ ID Nos 7 to 16, or having at least 85%, particularly at least 90%, more particularly at least 95%, even more particularly at least 99% sequence homology therewith.

5. Modified PAM or method according to any of the preceding claims, wherein the enzymaticactivity of PAM is the alpha-amidating activity of PAM.

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

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

7. Modified PAM or method according to any of the preceding claims, wherein the in vivohalf-life of said modified PAM, compared with unmodified PAM, is increased by at least 2-fold, particularly at least 5-fold, more particularly at least 10-fold, more particularly at least 20-fold, more particularly at least 50-fold, more particularly at least 100-fold, more particularly at least 150-fold, more particularly at least 250-fold, more particularly at least 350-fold, and / or wherein the in vivo half-life of said modified PAM, compared with unmodified PAM, isincreased by at least 0.5 hours, more particularly at least 1 hour, more particularly at least 2 hours, more particularly at least 5 hours, more particularly at least 10 hours, more particularly at least 24 hours, more particularly at least 48 hours, more particularly at least 60 hours and / orwherein the in vivo half-life of said modified PAM is at least 1 hour, more particularly 2hours, more particularly at least 5 hours, more particularly at least 10 hours, more particularly at least 24 hours, more particularly at least 48 hours, more particularly at least 60 hours.

8. Modified PAM or method according to any of the preceding claims, wherein the level ofPAM is increased in a sample obtained from a subject having received at least one dose ofsaid modified PAM, compared to a pre-dose sample obtained from said subject beforehaving received said at least one dose of said modified PAM, wherein said sample is obtained from said subject 2 hours, particularly 5 hours, more particularly 10 hours, more particularly 24 hours, more particularly 48 hours, more particularly 72 hours, more particularly 96 hours, more particularly 120 hours, more particularly 144 hours, more particularly 168 hours after administration of the last dose of said modified PAM to saidsubject.

9. Modified PAM or method according to any of the preceding claims, wherein at least 50%,particularly at least 75%, particularly at least 85%, particularly at least 95%, particularly about 100% of the enzymatic activity is maintained, compared to unmodified PAM.

10. Modified PAM or method according to any of the preceding claims, wherein said modifiedPAM, when administered to a subject, increases the bioavailability of one or more amidated peptides, in particular one or more peptide hormones in said subject.

11. Pharmaceutical composition comprising a modified PAM according to any of claims 1 to10.

12. Pharmaceutical composition according to claim 11, which is a ready-to-use formulation.

13. Pharmaceutical composition according to any of claims 11 to 12, which is a freeze-driedcomposition.

14. Modified PAM according to any of claims 1 to 10 or pharmaceutical composition accordingto any of claims 11 to 13 for effecting an increase in the level of PAM and / or the level ofalpha-amidating activity in the blood circulation of a subject, particularly by at least 2-fold, particularly at least 3-fold, particularly at least 5-fold, particularly at least 9-fold, particularly at least 10-fold, particularly wherein said increase lasts for at least 2 hours, more particularly at least 5 hours, more particularly at least 10 hours, more particularly at least 24 hours, more particularly at least 48 hours, more particularly at least 72 hours, more particularly at least 96 hours, more particularly at least 120 hours, more particularly at least144 hours, more particularly at least 168 hours.

15. Modified PAM according to any of claims 1 to 10 or pharmaceutical composition accordingto any of claims 11 to 13 for use as a medicament, particularly for use in the treatment of adisease or disorder selected from the group consisting of cardiovascular, kidney, inflammatory, infectious, and metabolic diseases or disorders, dementia, cancer, and other diseases or disorders connected with disturbed peptide homeostasis, wherein moreparticularly said disease or disorder is sepsis.

16. Modified PAM according to any of claims 1 to 10 or pharmaceutical composition accordingto any of claims 11 to 13 for reducing the risk of a subject for suffering from a medicalcondition, and / or for reducing the occurrence of a disease or disorder in said subject, and / or for reducing the severity of a disease or disorder in said subject.

17. Modified PAM according to any of claims 1 to 10 or pharmaceutical composition accordingto any of claims 11 to 13 for use in treatment or prevention of a disease or medical conditionin a subject, wherein said disease or medical condition is associated with endothelial barrier dysfunction and / or blood-brain barrier dysfunction.

18. Modified PAM for use in treatment or prevention of a disease in a subject according toclaim 17, wherein said disease or medical condition is selected from the group consisting of cardiovascular, kidney, inflammatory, infectious, and metabolic diseases or disorders, dementia, cancer, and other diseases or medical conditions connected with disturbed peptidehomeostasis, wherein more particularly said disease or disorder is sepsis.

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