Compounds and methods for persistently increasing PAM

Abstract

This invention relates to increasing the level of peptidyl glycine α-amidyl monooxygenase (PAM) in human circulation as a long-acting drug and for treating subjects, wherein said treatment includes: reducing the likelihood or risk of disease or condition, and / or reducing the occurrence of disease or condition, and / or reducing the severity of disease or condition.

Description

Technical Field

[0001] This invention relates to compounds and methods for sustainably increasing peptidyl-glycine α-amidyl monooxygenase (PAM) in subjects compared to unmodified PAM, as further described herein, wherein the enzymatic activity of modified PAM is maintained compared to unmodified PAM. The invention also relates to corresponding medical applications and pharmaceutical compositions.

[0002] The present invention also relates to compounds and methods for increasing the concentration of PAM in a subject and / or increasing the in vivo half-life of PAM, particularly in a subject, and specifically for achieving a sustained in vivo elevation of certain mature C-terminal amidated peptides, especially bioactive peptide hormones, such as bioactive adrenomedullin (bio-ADM). Further embodiments of the invention are described below. Background Technology

[0003] Bioactive peptide hormones function as signal transduction molecules. Most bioactive peptide hormones are synthesized from larger, inactive precursor peptides (see examples). Figure 1During their biosynthesis, these peptides undergo several co-translational and post-translational modifications, including cleavage of the signal peptide, endopeptidation of the precursor peptide by specific endopeptidases primarily at basic residue pairs, removal of basic residues by carboxypeptidases, disulfide bond formation, and N-glycosylation and O-glycosylation (Eipper et al., 1993. Protein Science 2(4): 489-97). More than half of the known neuropeptides and endocrine peptides require additional modification steps to acquire their full biological activity, including the formation of a C-terminal α-amide group (Guembe et al., 1999. JHistochem Cytochem 47(5): 623-36). This final step in peptide hormone biosynthesis involves the action of the bifunctional enzyme peptidylglycine α-amidylmonoxylase (PAM). PAM specifically recognizes the C-terminal glycine residue in its substrates, cleaving glyoxylate from the C-terminal glycine residue in a two-step enzymatic reaction, leading to the formation of a C-terminal α-amidated peptide hormone, in which the resulting α-amide group originates from the cleaved C-terminal glycine (Prigge et al., 2004. Science 304(5672): 864-67). This amidation reaction occurs in the lumen of the secretory granule before the amidation product is exocytotic (Martinez and Treston 1996. Molecular and Cellular Endocrinol 123: 113-17). α-amidated peptides include, for example, adrenomedullin (ADM), substance P, vasopressin, neuropeptide Y, amylin, calcitonin, and neurokinin A. However, it has previously been shown that PAM can also catalyze the formation of α-amides from non-peptide glycinate substrates, such as N-fatty acyl-glycine, which is converted by PAM to primary fatty acid amides (PFAMs), such as oleamide. The identified and purified peptidyl-glycine amidation activity was determined 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).

[0004] In humans, the PAM gene is located on chromosome 5q21.1, is 160 kb in length, and contains 25 known exons (Gaier et al., 2014. BMC Endocrine Disorders 14). It is known to produce at least 6 isoforms (SEQ ID 1-6) through alternative splicing. PAM enzymes have been found to be expressed at varying levels in almost all mammalian cell types, with significant expression in airway epithelium, endothelial cells, ependymal cells, adult atria, brain, kidneys, pituitary gland, gastrointestinal tract, and reproductive tissues (Chen et al., 2018. Diabetes Obes Metab 20 Supplement 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).

[0005] The precursor protein (1-973 amino acids) of the largest known PAM isoform 1 (SEQ ID No. 1) encoded by PAM cDNA is described in Figure 2 The N-terminal signal sequence or precursor peptide (amino acids 1-20) ensures the orientation of the nascent PAM polypeptide into the endoplasmic reticulum secretory lumen and is subsequently co-translated and cleaved. The PAM-propeptide is then processed by the same mechanisms used for the biosynthesis of intact membrane and secretory proteins, including the pre-cleavage region (amino acids 21-30), ensuring proper folding, disulfide bond formation, phosphorylation, and glycosylation (Bousquet-Moore et al., 2010. J Neurosci Res 88(12): 2535-45). Following cleavage of the N-terminal signal sequence or precursor peptide (amino acids 1-20) and the pro-region or propeptide (amino acids 21-30), the sequences of PAM isoforms 1-6 are given as SEQ ID Nos. 11-16.

[0006] like Figure 2As described, the PAM cDNA further encodes two distinct enzymatic activities. The first activity, named peptidyl-glycine α-hydroxylation monooxygenase (PHM; EC 1.14.17.3), is an enzyme that catalyzes the conversion of C-terminal glycine residues to α-hydroxy-glycine. The second activity, named peptidyl-α-hydroxy-glycine α-amidation lyase (PAL; EC 4.3.2.5), is an enzyme that catalyzes the conversion of α-hydroxy-glycine to α-amides, subsequently releasing glyoxylate. The sequential action of these individual enzymatic activities results in overall peptidyl-glycine α-amidation activity. The first enzymatic activity (PHM) is located directly upstream of the pre-region (amino acids 31-494 of isoform 1 (SEQ ID No. 7)). The second catalytic activity (PAL) is located within amino acids 495-817 (SEQ ID No. 8) after exon 16 in isoform 1.

[0007] like Figure 2As described, these two activities can be encoded together within a single polypeptide as membrane-bound proteins (isoforms 1, 2, 5, and 6; corresponding to SEQ ID Nos. 1, 2, 5, and 6 of prepro-PAM or SEQ ID Nos. 11, 12, 15, and 16 of PAM after cleavage of the prepro sequence) and within a single polypeptide as soluble proteins lacking a transmembrane domain (TMD) (isoforms 3 and 4; corresponding to SEQ ID Nos. 3 and 4 of prepro-PAM or SEQ ID Nos. 13 and 14 of PAM after cleavage of the prepro sequence). Although isoforms 1, 2, 5, and 6 remain in the extraplasmic membrane after fusion of secretory vesicles with the plasma membrane and subsequently undergo endocytosis and recycling or degradation, the TMD-deficient soluble PAM isoforms (isoforms 3 and 4) (amino acids 864-887) are co-secreted with the peptide hormone (Wand et al., 1985 Metabolism 34(11): 1044-52). Furthermore, in the secretory pathway, prohormone convertase can convert membrane-bound PAM proteins into soluble PAM proteins via cleavage within the flexible region (exon 25 / 26) connecting PAL and TMD (Bousquet-Moore et al., 2010. J Neurosci Res 88(12):2535-45). In the secretory pathway, PHM subunits can be cleaved from soluble or membrane-bound PAM by prohormone convertase acting on a bibasic cleavage site in exon 16. Additionally, during endocytosis, full-length PAM proteins may also be converted to soluble forms due to the action of α-secretase and γ-secretase (Bousquet-Moore et al., 2010. J Neurosci Res 88(12): 2535-45). Membrane-bound PAM from late endosomes can also be secreted in the form of exosomal vesicles.

[0008] The activities of PHM and PAL, as well as the activity of full-length PAM, were determined in several human tissues and body fluids. However, when individual reactions are permitted in the same compartment, body fluid, or in vitro experimental setup, the individual activities of soluble PHM and PAL also result in the formation of C-terminal α-amidation products from C-terminal glycinated substrates. To date, the transfer of PHM hydroxylation products to PAL is not fully understood. There is evidence that the hydroxylation products are released into solution rather than transferred directly from PHM to PAL (Yin et al., 2011. PLoS One 6(12): e28679). Furthermore, the origin of circulating PAM remains unclear.

[0009] Figure 3The paper describes part of the PHM reaction. PHM is a copper-dependent monooxygenase responsible for the stereospecific hydroxylation of C-terminal glycine at the α-carbon atom. In the hydroxylation reaction, ascorbic acid is considered to be a naturally occurring reducing agent, and the oxygen in the newly formed hydroxyl group has been shown to originate from molecular oxygen. Figure 3 The paper describes some of the reactions of PAL.

[0010] The catalytic action of PAM involves: the extraction of protons from hydroxyglycine formed by PHM via a protein backbone-derived base, and the nucleophilic attack of divalent metals by the oxygen in the hydroxyl group, leading to the cleavage of glyoxylate and the formation of a C-terminal amide. PAM activity in several human tissues and fluids from healthy samples and samples with several diseases was analyzed. Changes in PAM activity have been shown to be associated with different pathologies. Previous work is summarized below: Wand and colleagues demonstrated amidation activity in human cerebrospinal fluid (CSF) (Wand et al., 1985 Neuroendocrinol 41: 482-89). In patients with Alzheimer's disease (AD), PAM activity in CSF was significantly reduced compared to normal samples (Wand et al., 1987 Neurology 37: 1057-61). Furthermore, as reported in WO2015 / 103594, mass spectrometry analysis showed a reduction in PAM protein in the CSF of AD patients compared to healthy controls. Additionally, bio-ADM, as one of the PAM amidation products, has been shown to be reduced in patients with both epidemiological and sporadic Alzheimer's disease (WO2019 / 154900).

[0011] Amidation activity in the serum of patients with low back pain was analyzed using 1–12 P substance Gly (SP Gly) as a substrate (Hyyppä et al., 1990 Pain 43: 163–68). It has been shown that PAM activity is significantly reduced in serum of patients with multiple sclerosis (MS) (Tsukamoto et al., 1995. Internal Medicine 34(4): 229–32; WO2010 / 005387). The association between reduced plasma PAM activity and type 2 diabetes was described in (WO2014 / 118634). Furthermore, several PAM mutations leading to reduced activity have been shown to be associated with an increased risk of type 2 diabetes, possibly by affecting insulin particle packaging and release from β cells (Thomsen et al., 2018 Nat. Genet. 50: 1122-1131; Chen et al., 2020: Diabetologia 63: 561-576; Sheng et al., 2022 Funct. Integr. Genomics22: 525-535).

[0012] Compared with healthy controls, patients with type 1 multiple endocrine tumors (MEN-1) and pernicious anemia showed decreased plasma PAM activity (Kapuscinski et al., 1993. Clin Endocrinol 39(1): 51-58).

[0013] Increased PAM activity has been observed in infectious diseases such as sepsis and septic shock (Example 1). Specifically, in the prospective observational multinational adrenomedullin versus sepsis and septic shock-1 cohort (AdrenOSS-1), this increase in PAM activity predicted 28-day mortality with a hazard ratio (HR) of 2.11 (p=0.008) (Example 1).

[0014] Further analysis of the inactive ADM precursor adrenomedullin-glycine (ADM-Gly) showed significantly higher ADM-Gly concentrations in non-survivors compared to survivors (p<0.0001) (Example 2). Similarly, in this analysis, high ADM-Gly concentrations strongly predicted 28-day mortality, with a HR of 3.28 (p<0.0001) (Example 2). These findings suggest that PAM administration may be beneficial for patients with acute pathological conditions, particularly in cases requiring the conversion of inactive prohormone into active peptide hormones. The examples above demonstrate that changes in PAM activity are closely associated with the progression of various diseases.

[0015] Therefore, restoring PAM levels from the pathological state to those observed in the healthy state may reverse the pathological state. Thus, there is a need to develop methods to achieve a sustained increase in circulating PAM levels in the human body. Intravenous administration of recombinant natural PAM has been shown to increase PAM activity, leading to an increase in bio-ADM concentration in the bloodstream. However, due to the short average half-life of the natural enzyme in circulation (47 minutes), the duration of this effect is relatively short (Kaufmann et al., 2021, Scientific Reports (11): 1579).

[0016] As mentioned above, PAM is the only known enzyme that converts inactive hormones to their active forms via C-terminal α-amidation (Eipper et al., Annu Rev Neurosci 1992, 15: 57-85). More than half of known hormones require this process for activation. Rather than increasing the level of each peptide individually using different stabilization methods, increasing the circulating half-life of PAM provides a way to increase the levels of several amidated peptides. This could offer significant benefits in terms of treatment cost and efficiency, as well as potentially improving patient outcomes by increasing the availability of active peptides. Attached Figure Description

[0017] Figure 1 Schematic diagram of the adrenomedullin processing pathway. The N-terminal 20-peptide of proadrenomedullin (PAMP), the intermediate region of proadrenomedullin (MR-proADM), the C-terminal glycine-containing adrenomedullin (ADM-Gly), and the C-terminal proadrenomedullin (CT-proADM) are cleaved from the ADM precursor peptide proadrenomedullin (proADM). The final step of ADM activation is catalyzed by peptidyl-glycine α-amidyl-monooxygenase (PAM).

[0018] Figure 2 Schematic diagram of PAM isoform 1. The thick black arrows indicate the cleavage sites of the dibasic amino acids.

[0019] Figure 3 : A representative enzymatic reaction catalyzed by PAM.

[0020] Figure 4PAM activity in sepsis and septic shock. PAM activity in the AdrenOSS-1 subcohort. (A) Differences in PAM activity in Li-heparinized plasma measured at ICU admission in patients with sepsis and septic shock, analyzed by univariate ANOVA (Dunnehmann correction), with the control cohort including self-reported healthy individuals. Significance of differences is shown in each subplot. (b) 28-day Kaplan-Meil survival curves for low versus high PAM activity, based on a PAM activity cutoff of 35.1 units / L in patients with sepsis and septic shock.

[0021] Figure 5 Determination of ADM-Gly concentrations in sepsis and septic shock. ADM-Gly in the AdrenOSS-1 subcohort. (A) Differences in ADM-Gly concentrations in Li-heparinized plasma measured at ICU admission in patients with sepsis and septic shock, analyzed by one-way ANOVA (Dunne-corrected), with the control cohort including self-reported healthy individuals. Significance of differences is shown in each subplot. (b) 28-day Kaplan-Meyer survival curves: low ADM-Gly concentrations versus high ADM-Gly concentrations, with a cutoff of 730 pg / ml in patients with sepsis and septic shock.

[0022] Figure 6 Representative calibration curves of recombinant PAM (ADM maturation activity [AMA]).

[0023] Figure 7 AMA frequency distribution (histogram) in self-reported healthy individuals (n=120).

[0024] Figure 8 Characterization of PEGylated PAM using size exclusion chromatography and SDS-PAGE analysis.

[0025] 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.

[0026] Figure 10 Single-dose PAM injection. Comparison of PEG-PAM with unmodified PAM. Comparison of half-life of PEG-PAM with unmodified PAM: A single intravenous injection of 14.4 units / kg animal body weight was administered, and blood samples were collected within 300 minutes after administration for AMA analysis.

[0027] Figure 11Enrichment of plasma AMA after subcutaneous PEG-PAM injection: By measuring AMA within 24 hours after administration, an increase of 984% in circulating amidation activity was observed compared with baseline AMA.

[0028] Figure 12 Enrichment of plasma AMA after intraperitoneal injection of PEG-PAM: By measuring AMA within 8 hours after administration, an increase of 1900% in circulating amidation activity was observed compared with baseline AMA.

[0029] Figure 13 Enrichment of plasma AMA after intramuscular injection of PEG-PAM: By measuring AMA within 4 hours after administration, an increase of 1680% in circulating amidation activity was observed compared with baseline AMA.

[0030] Figure 14 Protective effect of PEG-PAM on blood-brain barrier integrity in a rodent sepsis model: The concentration of Evans blue dye in brain homogenate was used as a measure of blood-brain barrier integrity. Black bars indicate animals treated with 1×PBS (placebo group). Gray bars indicate animals treated with PEG-PAM. Significance was determined using a standard one-way ANOVA test with GraphPad Prism version 7.0; ns indicates no significant difference.

[0031] Figure 15 Left: Comparative activity analysis of PEGylated or Xtenized PAM enzymes and unmodified PAM enzymes. Right: Comparative analysis of PEGylated or Xtenized PAM enzymes and unmodified PAM enzymes on SDS-PAGE. Summary of the Invention

[0032] The subject of this invention is a method and compounds for achieving a sustained, non-toxic increase in PAM levels and / or amidated peptide levels in the blood circulation through modified PAM.

[0033] The term “PAM level” specifically refers to PAM activity and / or PAM concentration, and more particularly PAM activity, which in some embodiments is determined by measuring PAM amidation activity or by the PAM immunoassay described below.

[0034] In this invention, the amidation activity of the modified PAM is maintained; in particular, at least 10% amidation activity is maintained compared with 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%.

[0035] The terms “amidation activity,” “α-amidation activity,” “peptidyl glycine α-amidation activity,” “AMA,” or “PAM activity” refer to the sequential enzymatic activity of PHM and PAL, independent of the presence of splice variants or mixtures of splice variants, or post-translational modified PAM enzymes, or soluble isolated PHM or PAL activity, or soluble PHM and membrane-bound PAL, or combinations of all mentioned forms, resulting in the formation of α-amidated products of peptide or non-peptide-characteristic, particularly peptide-characteristic, glycinated substrates. In other words, the terms “amidation activity,” “α-amidation activity,” “peptidyl glycine α-amidation activity,” or “PAM activity” can be described as the sequential action of enzyme activity located within amino acids 21 to 817 of the original peptide encoded by human PAM cDNA, independent of the presence of splice variants or mixtures thereof.

[0036] The AMA of (modified) PAM can be determined, for example, in an aqueous solution (e.g., a buffer solution): a buffer solution containing an oxidase / peroxidase such as catalase, a protease inhibitor such as amoxicillin, leucine, a reducing agent such as ascorbate, and ADM-Gly or another suitable α-Gly peptide and an antibody against ADM-Gly or said α-Gly peptide is added, and a second solution (e.g., a solution containing a chelating agent such as EDTA) is added to fractions of the reaction solution at different time points to terminate the amidation reaction; then, the reaction products generated at different time points can be quantified, for example, by a quantitative immunoassay. A specific such assay is described herein in Example 3.

[0037] In some embodiments, AMA can also be determined in vitro by measuring the concentration of an amidated (e.g., synthetic) peptide with a C-terminal glycine using a detection method selected from, but not limited to, radioimmunoassay (RIA), homogeneous enzyme-multiplying immunoassay (EMIT), chemiluminescence and fluorescence immunoassay, enzyme-linked immunosorbent assay (ELISA), Luminex-based bead arrays, protein microarray assays, and rapid assay formats such as immunochromatographic strip assays.

[0038] PAM concentration can also be determined using PAM immunoassay, such as the following assays: - One-step method: Pipe the sample / calibrator (especially at least 10 μL) into a pre-coated container (especially a microtiter plate); add (especially 200 μL) of buffer containing labeled anti-PAM antibody (especially 300 mmol / L potassium phosphate, 100 mmol / L NaCl, 10 mmol / L Na-EDTA, 50 μmol / L amoxicillin, 100 μmol / L leucine, 0.1% bovine IgG, 0.02% mouse IgG, 0.5% BSA, pH 7.0), and incubate the microtiter plate (especially at room temperature (20°C) with stirring at 600 rpm for at least 3 hours); remove unbound tracers by washing (especially 5 times, 350 μL / well) with washing solution (especially 20 mmol / L PBS, 1 g / L Triton X-100, pH 7.4); measure the chemiluminescence of well-bound tracers, especially for 1 second per well, for example by using Centro LB. 960 microtiter plate luminescent reader (Berthold Technologies).

[0039] - Two-step method: Pipe the sample / calibrator (especially at least 10 μL) into a pre-coated container (especially a microtiter plate); add (especially 200 μL) buffer (as described in the one-step method), and incubate the microtiter plate (especially at 2 to 8 °C with stirring at 600 rpm for at least 15 to 20 hours); remove unbound sample by washing with washing solution (especially 4 times, 350 μL / well each time), then add (especially 200 μL) tracer and incubate the microtiter plate (especially at room temperature (20 °C) for 2 hours); remove unbound tracer by washing with washing solution (especially 4 times, 350 μL / well each time); measure the chemiluminescence of well-bound samples (especially 1 second per well, using a Centro LB 960 microtiter plate luminescence reader (Berthold Technologies)).

[0040] - The specific technology used is a sandwich luminescent immunoassay based on acridinium ester labeling; - The labeled compound (tracer) is specifically: a purified anti-PAM antibody (0.2 g / L) is labeled (particularly by incubation in 10% labeling buffer (particularly 500 mmol / L sodium phosphate, pH 8.0) with (particularly 1 g / L MACN-acridonium-NHS-ester, in particular 1:5 mol / L ratio, InVent Ltd.), particularly at 22°C for 20 min); the corresponding antibody is separated from the free label after the addition of buffer (particularly 5% 1 mol / L Tris-HCl, pH 8.0, 10 min) (particularly via a CentriPure P10 column (EMP Biotech Ltd.)); the purified labeled antibody is diluted (particularly in 300 mmol / L potassium phosphate, 100 mmol / L NaCl, 10 mmol / L Na-EDTA, 5 g / L bovine serum albumin (pH 7.0)); the final concentration is particularly 150 g / L per 150 g / L. μL of approximately 20 ng of labeled antibody.

[0041] - Solid phase: Coat (especially at 20°C for 18 hours) containers (especially white polystyrene microtiter plates (Greiner Bio-One International AG)) with buffer containing the corresponding antibody (especially 2 μg / 0.2 mL of 50 mmol / L Tris-HCl, 100 mmol / L NaCl, pH 7.8 per well); after blocking (especially with 30 g / L Karion, 5 g / L LBSA (protease-free), 6.5 mmol / L potassium dihydrogen phosphate, 3.5 mmol / L sodium dihydrogen phosphate (pH 6.5)), vacuum dry the plates.

[0042] - Calibration: The determination was calibrated using a commercially available dilution of recombinant PAM (particularly according to variant B of Example 4). Typical concentration ranges are from 1 to 1,000 ng / mL.

[0043] As used herein, PAM, particularly human PAM, more specifically includes all its isoforms and individual subunits, especially those produced by alternative splicing, and particularly proteins containing polypeptide chains of SEQ ID No. 1-10.

[0044] It will be apparent to those skilled in the art that SEQ ID No: 1 and SEQ ID No: 2-6, as shown in the corresponding sequence descriptions below, are the prepro form of PAM (containing a precursor peptide and a pro-peptide), while SEQ ID No. 10 relates to a pro-form fragment of PAM. Those skilled in the art can readily discern which portions of the prepro or pro-form sequence of PAM belong to the signal peptide or pro-peptide, and which belong to the mature PAM. Nevertheless, for completeness, the corresponding mature forms of PAM, i.e., the preferred PAM forms of the present invention, are shown in SEQ ID No: 11-16.

[0045] For example, according to the present invention, PAM can refer to PAM (SEQ ID No: 11) and / or fragments and / or isoforms (SEQ ID No: 12-16, SEQ ID No: 18), and / or catalytic subunits such as PHM (SEQ ID No: 7, 9) and PAL (SEQ ID No: 8), and / or fragments of them in unmodified or modified forms. With necessary modifications, this also applies to other embodiments of the present invention.

[0046] SEQ ID No. 10 is the wild-type pre-fraction of PAM, with a C-terminal amino acid extension sequence containing two amino acids, G (glycine) and S (serine). SEQ ID No. 18 is the wild-type fragment of PAM, with a C-terminal amino acid extension sequence containing two amino acids, G (glycine) and S (serine).

[0047] In an embodiment of the present invention, PAM is the recombinant PAM protein according to SEQ ID No. 17.

[0048] The term "unmodified PAM" refers to all isoforms and individual subunits of PAM, particularly those based on SEQ ID Nos. 1-10, which have not undergone any deliberate or artificial modification, including chemical conjugation, amino acid sequence alteration (including amino acid exchange, deletion, or insertion), fusion with other proteins, or other such methods. Although some variations may exist depending on the source organism, expression and / or purification conditions, and certain naturally occurring mutants and variants known in the art, unmodified PAM, in particular, possesses the amino acid sequence of naturally occurring PAM, and more particularly, at least substantially retains its native three-dimensional structure, function, and properties.

[0049] Unmodified PAM can also refer to recombinant PAM (SEQ ID No: 17).

[0050] The term "modified PAM" refers to all isoforms and individual subunits of PAM, particularly those based on SEQ ID Nos. 1-10, which are modified according to the invention, particularly as detailed herein, and more particularly by amino acid manipulation, by fusion with other proteins such as albumin (e.g., serum albumin or recombinant serum albumin), by non-covalent binding of serum albumin via a conjugated fatty acid chain linked to PAM, by fusion with the IgG Fc region or transferrin, and by post-translational modification by linking a natural or synthetic polymer, wherein the natural or synthetic polymer to be used is particularly HAP, more particularly ELP, more particularly PAS, more particularly PSA, more particularly GLK, more particularly XTEN, and even more particularly PEG.

[0051] The modified PAM can also refer to recombinant PAM (SEQ ID No: 17), which is modified by means of amino acid manipulation, preferably by fusion with albumin such as serum albumin or recombinant serum albumin, more preferably by non-covalent binding to serum albumin by linking to the conjugated fatty acid chain of PAM, more preferably by fusion with IgG Fc region or transferrin, and most preferably by post-translational modification by linking a natural or synthetic polymer, wherein the natural or synthetic polymer to be used is HAP, preferably ELP, more preferably PAS, more preferably PSA, more preferably GLK, more preferably XTEN, and most preferably PEG.

[0052] In some embodiments of the invention, the modified PAM or a pharmaceutical composition containing the modified PAM can be administered orally, epidermally, subcutaneously, intradermally, transdermally, sublingually, intramuscularly, intra-arterially, intravenously, or via the central nervous system (CNS, intracerebral, intraventricular, intrathecal), or via the nose or peritoneum. Specific routes of administration in this invention are epidermally, subcutaneously, intradermally, intramuscularly, intraperitoneally, and intravenously, more particularly epidermally, subcutaneously, intradermally, intramuscularly, and intraperitoneally, and even more particularly subcutaneously, intramuscularly, and intraperitoneally.

[0053] The in vivo half-life of modified and / or unmodified PAMs, as used herein, is specifically the time it takes for the level of (modified) PAM, particularly the α-amidation activity of (modified) PAM, to halve after reaching its maximum value following administration to a subject. The in vivo half-life of modified PAM can be determined, for example, by administering (e.g., by injection) the modified PAM to a test subject (e.g., an animal), determining the PAM amidation activity in samples collected at different time points before and after administration as described herein, plotting the results, and determining the half-life from the resulting curve; specifically using a method similar to that described in Example 3. With routine adjustments, these methods can be applied to other types of modified PAMs.

[0054] In one specific embodiment, the increase in half-life of modified PAM means that, compared with the half-life of unmodified PAM, the half-life after reaching its maximum value in cycling is increased by at least two times (i.e., 2 times), more particularly at least five times, more particularly at least ten times, more particularly at least 20 times, more particularly at least 50 times, more particularly at least 100 times, even more particularly at least 125 times, more particularly at least 150 times. In another specific embodiment, the increase in the half-life of modified PAM refers to an increase in the half-life of the unmodified PAM, measured by a suitable assay (e.g., the assay described in Example 3), after reaching its maximum value during cycling, to 1.05 to 2 times, or 1.05 to 5 times, or 1.05 to 10 times, or 1.05 to 20 times, or 1.05 to 50 times, or 1.05 to 100 times, or 1.05 to 125 times, or 1.05 to 150 times.

[0055] In some embodiments, the PAM level in a sample obtained from a subject who has received at least one dose of the modified PAM is increased compared to a pre-dose sample obtained from the subject before receiving the at least one dose of the modified PAM, wherein the sample is obtained from the 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 the last dose of the modified PAM is administered, or alternatively wherein the sample is obtained from the 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 the last dose of the modified PAM is administered.

[0056] "Bioavailability" specifically refers to the level of a detectable compound (e.g., peptide) present in the subject's circulation over a certain period of time after administration of PAM, particularly modified PAM, including, in particular, elevated levels. Increased bioavailability specifically refers to a prolonged elevation in the level of the corresponding compound in the subject's circulation after administration of the PAM, particularly modified PAM. In a particular embodiment, a prolonged elevation in the level of such a compound in the subject's circulation means an elevation for at least 1 hour, more particularly at least 8 hours, more particularly at least 12 hours, more particularly at least 24 hours, more particularly at least 48 hours, or even more particularly at least 72 hours after administration, in a particular embodiment 1 to 12 hours, or 1 to 24 hours, or 1 to 48 hours, or 1 to 72 hours after administration.

[0057] As used herein, when describing the effects of PAM modification, such as its effect on the half-life in the cycle, they refer to the effects and changes compared to unmodified PAM, especially unmodified PAM of the same type, such as the same isoform.

[0058] In certain implementations, the modified PAM includes modifications selected from the group consisting of: Amino acid manipulation (often also referred to as site-directed mutagenesis) specifically involves the insertion, deletion, and / or exchange of one or more amino acids within the amino acid sequence of a PAM, particularly to reduce in vivo immunogenicity and / or proteolytic instability compared to unmodified PAM. Thus, in certain embodiments, by manipulating one or more amino acids, modified PAM with enhanced in vivo protease resistance compared to unmodified PAM is obtained. For example, this includes increasing the conformational stability of the PAM and / or eliminating protease cleavage sites within the PAM.

[0059] PAM is conjugated with serum proteins such as albumin or immunoglobulins, or portions thereof, to produce a fusion protein. In certain embodiments, the conjugation occurs at the N-terminus or C-terminus, i.e., the serum protein is conjugated at the N-terminus or C-terminus of the PAM. Conjugation is particularly selected from post-translational chemical conjugation or conjugation via genetic engineering techniques. Specifically, post-translational chemical conjugation includes conjugation via cross-linking agents such as glutaraldehyde, carbodiimide, or maleimide, or conjugation via a conjugated fatty acid chain linked to the PAM. Specifically, conjugation via genetic engineering includes conjugation by modifying the DNA sequence encoding PAM to incorporate a gene encoding a serum protein or a portion thereof; in certain embodiments, the resulting DNA encoding a fusion protein comprising PAM and serum protein can be expressed using methods known to those skilled in the art to obtain the fusion protein.

[0060] ○ PAM protein is fused with albumin, such as serum albumin from natural sources or recombinant serum albumin; ○ PAM is fused to the IgG Fc region, specifically selected from human IgG1-Fc, IgG2-Fc, IgG3-Fc, IgG4-Fc, mouse IgG1-Fc, IgG2a-Fc, IgG2b-Fc, IgG3-Fc, rat IgG1-Fc, IgG2a-Fc, IgG2b-Fc, IgG2c-Fc, rabbit IgG-Fc, and canine IgG-Fc, and more specifically human IgG1-Fc, IgG2-Fc, IgG3-Fc, and IgG4-Fc; ○ PAM is fused with transferrin.

[0061] Post-translational modification of PAM by linking one or more natural or synthetic polymers, particularly polymers selected from the following group: ○ PEG (polyethylene glycol), especially linear or branched (e.g., containing 1+n branches) PEG, particularly those with an average molecular weight (M). n PEG in the range of 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 more particularly 5 to 50 kDa, especially those selected from PEG5000, PEG10000, PEG20000, and PEG40000, where the numbers refer to the average molecular weight in Da. In particular, the PEG is covalently linked to PAM. In one specific embodiment, the PEG is a 5000 Da NHS-PEG covalently fused to PAM. The molecular weight of the PEG can be determined by well-known methods, such as gel permeation chromatography (also known as size exclusion chromatography) or mass spectrometry, and is usually specified by the manufacturer.

[0062] ○ An unstructured peptide XTEN, covalently fused to PAM. In one particular embodiment, XTEN is an 864-membered single amino acid sequence composed of the amino acids Ala, Glu, Gly, Pro, Ser, and Thr in a randomized manner. In some embodiments, the half-life of the PAM-XTEN fusion protein can be customized by shortening the XTEN sequence.

[0063] ○ PAS, or peptide polymer composed of the amino acids proline, alanine, and serine, contains 100-200, particularly 120-180, more particularly 130-170, or even more particularly about 150 PAS repeating units.

[0064] ○ An ELP (elastin-like polypeptide) composed of valine-proline-glycine-x-glycine repeating units (naturally found in elastin), wherein x is any amino acid other than proline; in a particular embodiment, the ELP comprises 150, 200, 250, 300, 400 or 500 to 550, 600, 750, 850, 950 or 1,000 amino acids; ○ HAP, which is a repeating sequence of glycine-rich (Gly4Ser)n polypeptide, where n is 100-200, particularly 120-180, particularly 130-170, particularly about 150; ○ GLK (gelatin-like fusion protein), (Gly-XY)n structure, wherein X and Y are independently any amino acid except cysteine, and n = 60 to 1500, particularly 100 to 1250, particularly 150 to 1000, particularly 250 to 750, particularly 400 to 600, particularly about 500; ○ Branched or linear polysaccharides that can be linked to the N-terminus and / or C-terminus and / or amino acid side chains (especially lysine side chains) of PAM, particularly by means of N-glycosylation, for example, in vitro or by selecting an appropriate expression host in cell culture, and particularly selected from the following group: Dextran, particularly dextran selected from dextran 40, dextran 70 and dextran 500, wherein the numbers represent the approximate molecular weight of the dextran in kilodaltons (kDa); the molecular weight of dextran can be determined by well-known methods such as gel permeation chromatography (also known as size exclusion chromatography) or mass spectrometry, and is usually indicated by the manufacturer; Hydroxyethyl polysaccharides (HES) are particularly selected from hydroxyethylamine, hydroxyethyl methacrylate, hydroxyethyl hydrazine, hydroxyethyl disulfide and hydroxyethyl phosphonate.

[0065] Proheparin (HEP); Hyaluronic acid (HA).

[0066] ○ PSA (polysialic acid).

[0067] As used in this article, “conjugation” specifically refers to the covalent connection of a compound or substance to a PAM protein.

[0068] In some embodiments, PEGylation of PAM (PEGylation) refers to the attachment of PEG to a lysine side chain as detailed herein, particularly in alkaline aqueous solutions, such as in buffer solutions, especially at pH values ​​of 7.75 to 9.25, more particularly 8.25 to 8.75, and more particularly about 8.5; particularly, the molar excess of the PEG polymer relative to PAM is 70 to 140 times, more particularly 80 to 130 times, and more particularly 90 to 120 times; in a particular embodiment, a method similar to that described in Example 5 is employed, which can be adapted by conventional methods for other PAM types and / or other PEG polymers. 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.

[0069] In other preferred embodiments, the PAM is modified with PEG (PEGylation), preferably with PEG (PEG 5000 to PEG 10,000) having an average molecular weight of 5-10 kDa, or more preferably with PEG (PEG 5000) having an average molecular weight of about 5 kDa. In other embodiments, PEG modification of PAM (PEGylation) should refer to PEG 5000 or 10000 as detailed herein linked to the serine or lysine side chain of PAM, more preferably PEG 10000 as detailed herein linked to the lysine side chain of PAM, or more preferably PEG 5000 as detailed herein linked to lysine or serine, and even more preferably linked to the serine side chain of PAM.

[0070] In other preferred embodiments, PAM is modified with XTEN as defined herein, preferably with XTEN having the sequence SEQ ID No: 19, more preferably with the lysine or serine side chain of PAM, even more preferably with the lysine side chain of PAM, or alternatively with the addition of a Cys amino acid to the C-terminus of PAM.

[0071] PEG-PAM can refer to recombinant PAM (SEQ ID No: 17) modified with 1+n molecules of polyethylene glycol (PEG), where n is an integer in the range of 0 to 100, the molecular weight of a PEG molecule is in the range of 1-100 kDa, and it is a straight-chain molecule or a branched molecule with b+1 branches, where b is an integer in the range of 0 to 20.

[0072] In one specific embodiment, PEG-PAM may refer to recombinant PAM modified with PEG-5000 (5 kDa PEG) or PEG-10000 (10 kDa PEG) (SEQ ID No: 17).

[0073] PAM modified by site-directed mutagenesis can refer to recombinant PAM (SEQ ID No: 17) with n+1 additional amino acid insertions and / or n+1 amino acid deletions and / or n+1 amino acid exchanges in a given amino acid sequence (SEQ ID No: 17), where n is an integer in the range of 0-100.

[0074] PAM modified by fusion with natural serum albumin or recombinant human serum albumin can refer to recombinant PAM (SEQ ID No: 17) fused with natural human serum albumin or recombinant human serum albumin at the N-terminus or C-terminus of PAM according to (SEQ ID No: 17).

[0075] PAM modified by fusion with IgG Fc region can refer to recombinant PAM fused with IgG1 Fc region at N-terminus or C-terminus according to (SEQ ID No: 17) (SEQ ID No: 17).

[0076] PAM modified by XTEN can refer to recombinant PAM (SEQ ID No: 17) that is fused to the N-terminus or C-terminus of PAM according to (SEQ ID No: 17) and / or fused to any surface-exposed amino acid of PAM according to (SEQ ID No: 17).

[0077] PAM modified by XTEN can refer to recombinant PAM (SEQ ID No: 17) which is fused to the N-terminus or C-terminus of PAM with the XTEN moiety (SEQ ID No: 19) according to (SEQ ID No: 17) and / or fused to any surface-exposed amino acid of PAM according to (SEQ ID No: 17).

[0078] In some embodiments, site-directed mutagenesis includes creating expression vectors encoding modified protein sequences at the gene level to alter the biochemical and biophysical properties of PAM. Specifically, it should refer to the targeted exchange of one or more amino acids within the amino acid region 30 to 817 of SEQ ID No. 1 with different amino acids.

[0079] As used herein, the term "medical condition" includes diseases, symptoms, and adverse events, and more particularly includes the diseases, symptoms, and adverse events detailed herein.

[0080] The blood-brain barrier (BBB) ​​is a complex, dynamic interface that transduces biomechanical and biochemical signals from the vascular system and the brain. It is responsible for maintaining brain homeostasis by regulating the exchange of water, ions, nutrients, metabolites, neurotransmitters, and other cells (e.g., leukocytes), while restricting the entry of potentially toxic xenobiotics from the bloodstream. The BBB is partially formed by highly specialized endothelial cells lining brain capillaries. Tight junctions formed by brain microvascular endothelial cells (BMECs) regulate paracellular transport, while transcellular transport is regulated by specialized transport proteins, pumps, and receptors. This barrier regulates transport by transducing signals from the vascular system and the central nervous system. Typically, the vascular endothelium is particularly impaired in several pathological conditions, such as, but not limited to, stroke, multiple sclerosis, epilepsy, dementias such as Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, sepsis and severe sepsis, experimental colitis, and kidney disease (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)).

[0081] Typically, amidated peptide hormones, such as those found in vascular endothelium, are regulators of the blood-brain barrier (BBB). Evidence suggests that amidated hormones, such as, but not limited to, bio-ADM, oxytocin, and vasoactive intestinal peptide (VIP), mediate BBB function and regulation and / or have beneficial effects on BBB regeneration during and / or after diseases involving BBB disruption (Momenabadi, S. et al., Neuromolecular Med. 22, 557-571 (2020); and Yang, J. et al., Stroke Cerebrovasc. Dis. 31, 106160 (2022)). In addition, administration of PACAP slowed down AD-like lesions in mice, administration of VIP reduced amyloid β-plaque formation in 5×FAD transgenic mice, and prevented brain atrophy (Korkmaz, OT et al., J. Mol. Neurosci. 68, 389-396 (2019); and Rat, D. et al., FASEB J. 25, 3208 (2011)).

[0082] In one embodiment of the invention, in the clinical condition described above, the levels of circulating bio-ADM and other amidated peptides (such as, but not limited to, VIP and PACAP) are increased, thereby particularly promoting endothelial barrier regeneration and / or preventing endothelial barrier damage.

[0083] Little is known about the role or impact of peptide alpha-amidation (PAM) in clinical diseases. Wand et al. provided preliminary evidence of alpha-amidation activity in human circulation (Wand, GS, Ney, RL et al., Characterization of peptide alpha-amidation activity in humancerebrospinal fluid and central nervous system tissue, Neuroendocrinology 41, 482-489 (1985)). They reported no sex difference in PAM activity, but some variations were observed in certain disease states: plasma PAM activity was increased in hypothyroid adults and patients with medullary thyroid carcinoma. Elevated PAM activity was observed in medullary thyroid carcinoma, pheochromocytomas, and pancreatic islet tumor tissues, indicating increased formation of amidated peptides in endocrine tumor tissues (Gether, U., Aakerlund, L., and Schwartz, TW et al., Comparison of peptidyl-glycine α-amidation activity in medullary thyroid carcinoma cells, pheochromocytomas, and serum, Mol. Cell. Endocrinol. 79, 53-63 (1991); and Wand, GS, Nev, RL, Mains, RE, and Eipper, BA et al., Characterization of Peptide Alpha-Amidation Activity in Human Cerebrospinal Fluid and Central Nervous System Tissue, Neuroendocronology 41, 482-489 (1985). Patients with type 1 multiple endocrine tumors (MEN-1) and pernicious anemia showed reduced plasma PAM activity compared to healthy controls. Wand and colleagues demonstrated amidation activity in human cerebrospinal fluid (CSF).In patients with Alzheimer's disease (AD), plasma PAM activity was shown to be unchanged compared to healthy controls, while CSF PAM activity was significantly reduced compared to normal samples (Wand, GS et al., Alzheimer's disease: Low levels of peptidea-amidation activity in brain and CSF. Neurology 37, 1057-1061 (1987)). Furthermore, WO2015 / 103594 reported that mass spectrometry analysis showed reduced PAM protein levels in the CSF of AD patients compared to healthy controls. Additionally, bio-ADM, as one of the hormones involved in PAM amidation, has been shown to be reduced in patients with both epidemiological and sporadic Alzheimer's disease (WO2019 / 154900). According to reports in WO2021170816A1 and WO2021170752A1, circulating PAM activity is directly associated with the prediction, diagnosis, or progression of AD. PAM activity has been shown to be increased in CSF and significantly decreased in serum in patients with multiple sclerosis (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). The association between plasma PAM activity and type 2 diabetes is described in (WO2014 / 118634). Furthermore, WO2021170816A1 and WO2021170752A1 indicate increased circulating PAM activity due to acute illnesses such as sepsis and shock. To date, there are no reports of PAM undergoing clinical trials as a candidate drug.

[0084] The blood-brain barrier (BBB) ​​is a continuous endothelial membrane within brain microvessels, providing sealed intercellular contacts and being covered by wall vascular cells and perivascular astrocyte terminale. Extensive literature evidence indicates that bio-ADM is indispensable for intact endothelial and BBB function, and that administration of bio-ADM and other peptide hormones to supraphysiological levels has a protective effect on endothelial and BBB function (see review). Kis B et al., Peptides. January 2006; 27(1):211- 22The brain's bloodstream (BBB) ​​protects against the influence of factors present in the neuronal receptor cycle and maintains the highly regulated CNS environment required for normal synaptic and neuronal function. BBB disruption leads to the influx of neurotoxic hematogenous debris, cellular and microbial pathogens into the brain and is associated with inflammatory and immune responses, thereby initiating multiple neurodegenerative pathways. Sweeney, MD, and others Nat. Rev. Neurol. 14, 133-150 (2018) BBB dysfunction occurs in several diseases, including MS, epilepsy, stroke, and dementia, including Alzheimer's disease (AD). In these disorders, BBB dysfunction plays a crucial role in the pathogenesis because BBB destruction leads to ion regulation disorders, edema, neuroinflammation, ultimately resulting in neuronal dysfunction, increased intracranial pressure, and neurodegenerative changes. Profaci, CP et al., Experimental Medicine, 217.4 (2020) ).

[0085] The pharmaceutical compositions according to the present invention can treat and / or prevent BBB dysfunction and regulation disorders, and are therefore used to treat and / or prevent diseases associated with BBB dysfunction and / or disruption, including but not limited to brain and nervous system diseases such as cerebral infarction, mild cognitive impairment, dementia, vascular dementia, Alzheimer's disease, and encephalitis.

[0086] Advanced clinical imaging techniques can be used to quantify BBB damage and / or destruction and / or increased permeability in patients, 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 labeling (ASL), and intravoxel incoherent motion imaging (IVIM). These advanced clinical imaging techniques can help identify patients with BBB impairment in clinical settings, including but not limited to those with acute stroke, cerebral small vessel disease, and dementia such as mild cognitive impairment (MCI) and Alzheimer's disease (AD). (See review:) Chassidim, Y. et al., Fluids Barriers CNS 10, 9 (2013) ;as well as Elschot EP et al. Invest Radiol. January 2021; 56(1) ).

[0087] The main component of the blood-brain barrier (BBB) ​​is the endothelium, which not only plays a specific role in BBB formation but also commonly participates in vascular barrier formation. Dysfunction of the blood-brain barrier and vascular barrier, or endothelial dysfunction, is a systemic pathological state of the endothelium and can be broadly defined as an imbalance between vasodilators and vasoconstrictors produced and / or acting on the endothelium. Deanfield J et al., J Hypertens. January 2005; 23(1):7-17The normal functions of endothelial cells include regulating coagulation, platelet adhesion, immune function, and controlling the volume and electrolyte content of the intravascular and extravascular spaces. The endothelium is a monolayer of cells lining the entire cardiovascular system and regulating many processes, including vascular tone, thrombosis, angiogenesis, and inflammation. Endothelial cells have been shown to be phenotypically dynamic and capable of transitioning between resting and activated states in response to a variety of local and systemic stimuli. Colombo PC et al., Curr Heart Fail Rep. 2015 Jun;12(3):215-22) Recent research has shown that endothelial dysfunction is a major contributing factor to cardiovascular diseases, including hypertension, atherosclerosis, and congestive heart failure. Gutiérrez E et al. Eur Heart J. November 2013;34(41):3175- 81) The endothelium precisely controls the exchange of fluids from the circulation to surrounding tissues, and dysfunction of this barrier leads to uncontrolled fluid extravasation, which can result in congestion and / or edema. A common feature of edema (e.g., pulmonary edema) is increased permeability to low molecular weight water solutes. Rocker GM et al., Thorax. August 1987;42(8):620-3) Endothelial dysfunction can originate from several disease processes, such as hypertension, hypercholesterolemia, diabetes, or septic shock, and / or exacerbate these diseases. Endothelial dysfunction is a major pathophysiological mechanism leading to coronary artery disease and other atherosclerotic diseases. Therefore, the pharmaceutical compositions of the present invention can be used to treat and / or prevent diseases associated with endothelial and / or vascular dysfunction and / or damage.

[0088] Therefore, a common feature of the above and the following diseases is endothelial barrier dysfunction, including but not limited to vascular endothelium, blood-brain barrier, and intestinal endothelial barrier. The symptoms, diseases, and / or conditions mentioned include, but are not limited to, the following: (1) Brain and nervous system diseases: cerebral infarction, mild cognitive impairment, dementia, vascular dementia, Alzheimer's disease and encephalitis.

[0089] (2) Infectious diseases caused by infectious organisms such as bacteria, viruses, fungi or parasites, especially those caused by infectious bacteria, particularly those selected from SIRS, sepsis and septic shock.

[0090] (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.

[0091] (4) Endocrine and metabolic disorders: diabetes and diabetic organ disorders (e.g., diabetic nephropathy or diabetic retinopathy).

[0092] (5) Cardiovascular diseases: heart failure, pulmonary hypertension, occlusive arteriosclerosis, Berger's disease, myocardial infarction, lymphedema, Kawasaki disease, myocarditis, arrhythmia (e.g., arrhythmia after catheter ablation), atrial fibrillation, aortitis, pulmonary hypertension, hypertension, organ damage caused by hypertension, peripheral vascular disease and arteriosclerosis.

[0093] (6) The brain / nervous system condition prevented or treated by the pharmaceutical composition according to one embodiment of the present invention is dementia, particularly mild cognitive impairment, cerebral infarction dementia or Alzheimer's disease.

[0094] Dementia is a clinical syndrome characterized by a range of symptoms and signs, including memory difficulties, language impairment, psychological and mental changes, and disturbances in daily living activities. The different causes of dementia syndromes (sometimes referred to as subtypes) are: Alzheimer's disease (approximately 50% of cases), vascular dementia (approximately 25%), mixed Alzheimer's disease and vascular dementia (included in the above category, 25%), Lewy body dementia (15%), and others (approximately 5% in total), including frontotemporal dementia, focal dementia (such as progressive aphasia), subcortical dementia (such as Parkinson's disease dementia), and secondary causes of dementia syndromes (such as intracranial lesions).

[0095] Alzheimer's disease (AD) is the most common form of dementia. Key molecular mechanisms and histopathological features in the AD brain include a dynamic cascade of biochemical events, including the pathological amyloid precursor protein (APP) cleavage and the production of various β-amyloid substances (including amyloid-β peptide (Aβ)). 1-42 (dimers, trimers, oligomers) and subsequent amyloid aggregation and deposition in plaques, abnormal hyperphosphorylation and aggregation of tau protein, progressive intracellular neurofibrillary degeneration, changes and inflammation within the innate immune system, and blood-brain barrier damage.

[0096] Mild cognitive impairment (MCI) is a heterogeneous clinical disorder with several potential etiologies. However, a large proportion of MCI represents a transitional state between healthy aging and very mild AD. DeCarli 2003. Lancet Neurol. 2: 15-21 Therefore, studies have shown that MCI subjects tend to progress to clinically suspected AD at a rate of approximately 10%-15% per year. Markesbery 2010. J Alzheimers Dis. 19:221-228 Blood-brain barrier disruption in the hippocampus of MCI patients adds to the hypothesis that BBB disruption precedes neurodegenerative disease.

[0097] In a preferred embodiment, the patient group for Alzheimer's disease can be determined by risk factors such as the incidence of MCI, the presence of the genetic risk factor ApoE4, and age. In a more preferred embodiment, the risk factor age for Alzheimer's disease patients is defined as at least 60 years old.

[0098] Lewy body dementia (DLB) is a type of dementia that worsens over time. DLB is associated with BBB dysfunction and microvascular complications. Janelidze S et al., Neurobiol Aging. March 2017;51:104-112 Other symptoms may include fluctuating alertness, visual hallucinations, bradykinesia, difficulty walking, and limb rigidity.

[0099] DLB is the most common cause of dementia after Alzheimer's disease and vascular dementia. It typically begins after age 50. Underlying mechanisms include the formation of Lewy bodies, composed of α-synuclein, in neurons. A suspected diagnosis can be made based on symptoms, while blood tests and medical imaging are performed to rule out other possible causes. Currently, there is no cure for DLB. (See review article for more details.) McKeith et al., 2017. Neurology 89: 88-100 .

[0100] Vascular dementia (VaD), also known as multi-infarct dementia (MID) and vascular cognitive impairment (VCI), is a type of dementia caused by problems with blood supply to the brain. It typically results from a series of minor strokes, leading to a gradual decline in cognitive function. The term refers to a syndrome comprised of the complex interaction of cerebrovascular disease and numerous risk factors that cause changes in brain structure due to stroke and lesions, resulting in disruption of the blood-brain barrier and cognitive impairment. The time relationship between stroke and cognitive deficit is required for diagnosis.

[0101] Frontotemporal dementia (FTD) is a clinical manifestation of degenerative changes in the frontotemporal lobe, characterized by progressive neuronal loss primarily affecting the frontal or temporal lobes. Spindle neurons typically lose more than 70% of their number, while other neuronal types remain intact. FTD is associated with BBB dysfunction and microvascular complications. Janelidze S et al., Neurobiol Aging. March 2017; 51: 104-112 It accounts for 20% of early-onset dementia cases. Signs and symptoms typically appear in late adulthood, more commonly between 55 and 65 years of age, with men and women being affected to roughly the same extent. Common signs and symptoms include significant changes in social and personal behavior, apathy, emotional blunting, and a dual deficit in expressive and perceptual language. Currently, there is no cure for FTD, but treatments are available to help alleviate symptoms. See review. Bott et al., 2014. Neurodegener Dis Manag 4 (6): 439-454 .

[0102] Differentiating between different dementia syndromes can be challenging because clinical features and associated underlying pathologies often overlap. In particular, Alzheimer's dementia frequently co-occurs with vascular dementia. Patients with vascular dementia exhibit progressive cognitive impairment, which occurs acutely or subacutely following multiple cerebrovascular events (strokes), and is typically gradual, as in mild cognitive impairment. See review. Venkat et al., 2015. Exp Neurol 272: 97-108 Nevertheless, damage to the blood-brain barrier is a common pathological feature in all forms of dementia.

[0103] As used in this article, “dementia” generally refers to a clinical syndrome characterized by a range of symptoms and signs, including memory difficulties, language dysfunction, psychological and mental changes, and impairment of daily living activities, as mentioned above, which are characterized by disruption of the BBB and / or impaired regulation.

[0104] Therefore, "dementia" can refer to frontotemporal dementia, vascular dementia, Lewy body dementia, Alzheimer's dementia, and mild cognitive impairment, and the pharmaceutical compositions according to the present invention can be used to treat and / or prevent dementia by preventing BBB dysregulation.

[0105] (7) The pharmaceutical composition according to one embodiment of the present invention is used to prevent or treat infectious diseases, particularly sepsis or septic shock.

[0106] Endothelial cells are active contributors to sepsis and therefore represent a primary therapeutic target. During sepsis, endothelial cells amplify the immune response and activate the coagulation system. These cells are both targets and sources of inflammation, and act as a link between local and systemic immune responses. In response to cytokines produced by immune cells, endothelial cells express adhesion molecules and produce vasoactive compounds, inflammatory cytokines, and chemokines. Dolmatova EV et al., Cardiovasc Res. January 1, 2021; 117(1):60-73 Hemodynamic instability plays a crucial role in the development of sepsis and / or septic shock, and occurs due to a combination of sepsis-induced vasodilation and vascular leakage, the latter stemming from impaired endothelial integrity. During sepsis, injury-associated and pathogen-associated molecular patterns in circulation activate inflammatory and coagulation pathways, leading to widespread changes in the endothelium. This subsequently results in increased leukocyte adhesion, a procoagulant state, vasodilation, and endothelial cell permeability, ultimately leading to widespread edema, shock, and fatal organ dysfunction. Geven C. et al., Shock. Dec 2018;50(6):648-654 As shown in Example 8, the pharmaceutical composition according to the present invention can be used to prevent endothelial barrier regulation disorders and / or prevent endothelial permeability.

[0107] (8) The pharmaceutical composition according to one embodiment of the invention prevents or treats gastrointestinal diseases, particularly inflammatory diseases (e.g., inflammatory bowel disease (IBD) or Crohn's disease), ulcerative diseases (e.g., ulcerative colitis) or intestinal Behcet's disease.

[0108] Inflammatory bowel disease (IBD) includes Crohn's disease (CD), ulcerative colitis (UC) (and indeterminate colitis), which share several inflammatory features with other chronic immune disorders, including immune activation, leukocyte infiltration of tissues, and increased vascular density. Maintaining a 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 and animal models of IBD.

[0109] This alteration in solute permeability in the vascular system is not limited to the intestinal microcirculation, but broadly affects the vascular systems of other organs, including the brain. Cromer WE et al., World Journal of Gastroenterol. February 7, 2011; 17 (5):578-93 As shown in Example 8, the pharmaceutical composition according to the present invention can be used to prevent vascular barrier dysfunction and / or prevent vascular permeability.

[0110] (9) Endocrine and metabolic disorders that are prevented or treated by the drug of this embodiment, particularly diabetes and diabetic organ disorders (e.g., diabetic nephropathy or diabetic retinopathy).

[0111] Type 2 diabetes treatment based on glucagon-like peptide-1 (GLP-1) involves the following medications: GLP-1 receptor agonists that stimulate the GLP-1 receptor, such as liraglutide, albiglutide, or taspoglutide; or dipeptidyl peptidase-4 (DPP-4) inhibitors that prevent the inactivation of endogenous GLP-1, thereby increasing the concentration of endogenous active GLP-1. GLP-1 and its analogues activate pancreatic receptors, improving blood glucose levels by glucose-dependently stimulating insulin secretion and inhibiting glucagon secretion. Based on rodent studies, they also possess potential β-cell protective effects. GLP-1 receptors are also expressed in extrapancreatic tissues, showing therapeutic potential for weight loss and possibly beneficial cardioprotective and endothelial protective effects. Clinical trials in subjects with type 2 diabetes have demonstrated the effectiveness of GLP-1 analogue therapy as monotherapy and in combination with metformin, sulfonylureas, thiazolidinediones, or insulin over periods exceeding 12 weeks. Furthermore, GLP-1 receptor agonists reduce weight, while DPP-4 inhibitors have no effect on weight. This treatment is safe, with a very low risk of adverse events, including hypoglycemia. GLP-1-based therapy is currently the accepted treatment for type 2 diabetes, and its combination with metformin has unique value for patients whose diabetes is not well controlled by metformin monotherapy. Ahrén B. Exp Cell Res. May 15, 2011;317(9):1239-45 GLP-1 is a peptide hormone that needs to be PAM-amided.

[0112] The pharmaceutical compositions according to the invention are preferably used for the prevention or treatment of the above-mentioned symptoms, diseases and / or conditions (e.g., cardiovascular diseases, brain and neurological disorders, or endocrine and gastrointestinal disorders).

[0113] In certain embodiments, the pharmaceutical composition according to the invention is preferably a medicament for the prevention or treatment of the aforementioned symptoms, diseases, and / or conditions (e.g., cardiovascular diseases, brain and neurological disorders, or endocrine and gastrointestinal disorders), and further comprises vitamin C. Similarly, embodiments of the invention relate to a therapeutic and / or preventive compound, combination, or method, which in certain embodiments includes the addition of vitamin C.

[0114] In embodiments of the present invention, the pharmaceutical composition is used to treat and / or prevent cardiovascular diseases such as heart failure, pulmonary hypertension, occlusive arteriosclerosis, Berger's disease, myocardial infarction, lymphedema, Kawasaki disease, myocarditis, arrhythmias (e.g., post-catheter arrhythmias), atrial fibrillation, aortitis, hypertension, organ damage caused by hypertension, peripheral vascular disease, and arteriosclerosis.

[0115] In a preferred embodiment of the present invention, the pharmaceutical composition is used to treat and / or prevent heart failure, pulmonary hypertension, hypertension, organ damage caused by hypertension, peripheral vascular disease, and arteriosclerosis.

[0116] In the most preferred embodiment of the present invention, the pharmaceutical composition is used to treat and / or prevent pulmonary hypertension and hypertension.

[0117] In other embodiments of the invention, the pharmaceutical composition is used to treat and / or prevent endocrine and metabolic disorders, such as diabetes and diabetic organ disorders (e.g., diabetic nephropathy or diabetic retinopathy).

[0118] In a preferred embodiment of the invention, the pharmaceutical composition is used to treat and / or prevent type 2 diabetes.

[0119] In other embodiments of the invention, the pharmaceutical composition is used to treat and / or prevent gastrointestinal diseases such as inflammatory diseases (e.g., inflammatory bowel disease or Crohn's disease), ulcerative diseases (e.g., ulcerative colitis), intestinal Behcet's disease, hepatitis, liver fibrosis, cirrhosis, and liver failure.

[0120] In a preferred embodiment of the invention, the pharmaceutical composition is used to treat and / or prevent inflammatory diseases such as inflammatory bowel disease or Crohn's disease, ulcerative diseases such as ulcerative colitis and / or intestinal Behcet's disease.

[0121] In the most preferred embodiment of the invention, the pharmaceutical composition is used to treat and / or prevent inflammatory bowel disease and / or Crohn's disease.

[0122] In other embodiments of the invention, the pharmaceutical composition is used to treat and / or prevent infectious diseases caused by infectious organisms such as bacteria, viruses, fungi or parasites, more particularly by infectious bacteria, especially diseases selected from SIRS, sepsis and septic shock.

[0123] In a preferred embodiment of the invention, the pharmaceutical composition is used to treat and / or prevent sepsis and / or septic shock.

[0124] In embodiments of the present invention, the pharmaceutical composition is used to treat and / or prevent brain and neurological diseases such as cerebral infarction, mild cognitive impairment, dementia, vascular dementia, Alzheimer's disease, and encephalitis.

[0125] In a preferred embodiment of the present invention, the pharmaceutical composition is used to treat and / or prevent cerebral infarction.

[0126] In a more preferred embodiment of the invention, the pharmaceutical composition is used to treat and / or prevent dementia, including mild cognitive impairment, vascular dementia, and Alzheimer's disease.

[0127] In the most preferred embodiment of the invention, the pharmaceutical composition of the invention is used to prevent and / or treat symptoms associated with mild cognitive impairment (MCI) and Alzheimer's disease (AD).

[0128] In some embodiments of the present invention, the pharmaceutical composition is used to treat and / or prevent pathological disorders associated with endothelial and / or blood-brain barrier disorders.

[0129] In a preferred embodiment of the invention, the pharmaceutical composition is used to treat and / or prevent endothelial and / or blood-brain barrier dysfunction associated with sepsis or septic shock.

[0130] Some embodiments of the present invention relate to the use of modified PAM for the treatment or prevention of diseases or medical conditions in subjects, wherein the diseases or medical conditions are associated with endothelial barrier dysfunction and / or blood-brain barrier dysfunction.

[0131] A more preferred embodiment of the present invention relates to modified PAM for treating or preventing disease in subjects, wherein the disease or medical condition is selected from cardiovascular, renal, inflammatory, infectious and metabolic diseases or conditions, dementia, cancer and other diseases or medical conditions associated with peptide homeostasis disorders, wherein more particularly, the disease or condition is sepsis.

[0132] In a more preferred embodiment of the invention, the pharmaceutical composition is used to treat and / or prevent endothelial and / or blood-brain barrier dysfunction associated with cerebral infarction.

[0133] In a more preferred embodiment of the invention, the pharmaceutical composition is used to treat and / or prevent endothelial and / or blood-brain barrier dysfunction associated with dementia, including mild cognitive impairment, vascular dementia, and Alzheimer's disease.

[0134] In the most preferred embodiment of the invention, the pharmaceutical composition is used to treat and / or prevent endothelial and / or blood-brain barrier dysfunction associated with mild cognitive impairment (MCI) and Alzheimer's disease (AD).

[0135] More preferably, the pharmaceutical composition according to the invention is used for the prevention or treatment of heart failure, acute myocardial infarction, arrhythmia, atrial fibrillation, pulmonary hypertension, peripheral vascular disease, stroke, dementia, inflammatory bowel disease, Crohn's disease, ulcerative colitis, intestinal Behcet's disease, diabetes, diabetic nephropathy, diabetic retinopathy, pulmonary fibrosis, sepsis, or septic shock.

[0136] As used herein, “prevention” means essentially preventing the occurrence (onset or manifestation) of symptoms, diseases, and / or conditions. Furthermore, as used herein, “treatment” means suppressing (e.g., inhibiting progression), alleviating, repairing, and / or curing symptoms, diseases, and / or conditions that have already occurred (onset or manifestation).

[0137] As used in this article, the term "amino acid" specifically refers to naturally occurring amino acids, especially those naturally occurring in animals, especially those naturally occurring in mammals, and especially the 20 typical amino acids.

[0138] In some embodiments, the medical condition is a disease or symptom characterized by peptide homeostasis disorder, characterized in that, in samples of bodily fluids or tissue extracts obtained from the subject, The levels of PAM and / or its isoforms and / or fragments (particularly proteins containing the amino acid sequences of SEQ ID No. 1 to 10) are predetermined, particularly below 24 units / L, more particularly below 20 units / L, more particularly below 16 units / L, more particularly below 14 units / L, most particularly below 10 units / L, and / or One or more peptides have a peptide-Gly / peptide-amide ratio greater than a predetermined threshold, wherein particularly the ratio is greater than 1, more particularly greater than 1.5, more particularly greater than 1.6, more particularly greater than 1.7, more particularly greater than 1.8, more particularly greater than 1.9, and even more particularly greater than 2, and wherein particularly, the peptide is independently selected from adrenomedullin, substance P, vasopressin, oxytocin, amylin, PAMP, α-MSH, calcitonin, kissing peptide, neuropeptide Y, vasoactive intestinal peptide, and thyrotropin-releasing hormone.

[0139] In some embodiments, the medical condition is a disease or symptom characterized by peptide homeostasis disorder, characterized in that, in samples of bodily fluids or tissue extracts obtained from the subject, The levels of PAM and / or its isoforms and / or fragments (particularly proteins containing the amino acid sequences of SEQ ID No. 1 to 10) are predetermined for PAM activity and are particularly below 24 units / L, more particularly below 20 units / L, more particularly below 16 units / L, more particularly below 14 units / L, most particularly below 10 units / L, and / or The level of PAM and / or its isoforms and / or fragments (particularly proteins containing the amino acid sequences of SEQ ID No. 1 to 10) is the total concentration of PAM, which is predetermined and 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, most preferably below 40 ng / mL, and / or One or more peptides have a peptide-Gly / peptide-amide ratio greater than a predetermined threshold, wherein particularly the ratio is greater than 1, more particularly greater than 1.5, more particularly greater than 1.6, more particularly greater than 1.7, more particularly greater than 1.8, more particularly greater than 1.9, and even more particularly greater than 2, and wherein particularly, the peptide is independently selected from adrenomedullin, substance P, vasopressin, oxytocin, amylin, PAMP, α-MSH, calcitonin, kissing peptide, neuropeptide Y, vasoactive intestinal peptide, and thyrotropin-releasing hormone.

[0140] In a particular implementation, a disease or condition characterized by peptide homeostasis disorder is characterized by the presence of peptides in a fluid or tissue extract sample obtained from the subject. The ADM-Gly / bio-ADM ratio is higher than a predetermined threshold, particularly higher than 1, more particularly higher than 1.5, more particularly higher than 1.6, more particularly higher than 1.7, more particularly higher than 1.8, more particularly higher than 1.9, or even more particularly higher than 2, and / or The bio-ADM concentration was below a predetermined threshold, specifically below 20 pg / mL, even more specifically below 15 pg / mL, even more specifically below 10 pg / mL, and even more specifically below 5 pg / mL.

[0141] In one embodiment, the subject's bodily fluids

[0142] - The molar ratio (peptide-Gly / peptide-amide) of the peptide-Gly to its corresponding peptide-amide (peptide-NH2) and / or

[0143] - PAM concentration and / or

[0144] - PAM activity

[0145] The test can be performed at least once before and / or at least once after the treatment of the subject.

[0146] In a more specific embodiment, the subject's bodily fluids

[0147] - The molar ratio of ADM-Gly to bio-ADM (ADM-Gly / bio-ADM) and / or

[0148] - concentration of bio-ADM and / or

[0149] - PAM concentration and / or

[0150] - PAM activity

[0151] The test can be performed at least once before and / or at least once after the treatment of the subject.

[0152] Those skilled in the art will understand that, after treatment of the subject with the PAM, the aforementioned PAM concentration or PAM activity comprises endogenous PAM and / or its isoforms and / or fragments thereof, as well as exogenous (therapeutic) PAM and / or its isoforms and / or fragments thereof, including recombinant PAM according to SEQ ID No: 17, whether modified or unmodified.

[0153] In some implementations, a threshold is predetermined by measuring the levels of PAM and / or its isoforms and / or fragments and / or ADM-Gly and / or bio-ADM in samples such as those obtained from a subject cohort, and calculating, for example, the 25th percentile, more particularly the 10th percentile, or even more particularly the 5th percentile, to define the threshold, thereby characterizing the subject as having a medical condition or at risk of having a medical condition.

[0154] As used herein, a subject cohort may refer to a representative sample of subjects from the general population, which is typically randomly selected; in some embodiments, this may be, for example, a research population from a medical study, and in particular embodiments, it may involve a target cohort, such as within a certain age range, sex, certain physical conditions, biomarkers (such as BMI), etc. In some specific embodiments, the subject cohort consists of asymptomatic subjects who appear to be healthy (especially self-reported healthy); some of these subjects may actually have underlying, currently undiagnosed medical conditions associated with reduced PAM levels.

[0155] In a particular embodiment, the levels of PAM and / or its isoforms and / or fragments are detected as total PAM concentration and / or PAM activity. Specifically, this refers to the total concentration of all PAM isoforms and the total α-amidation activity measurable in the sample.

[0156] In other embodiments, such as those determined using a PAM activity assay (particularly the PAM activity assay described herein), a specific threshold for PAM activity in samples (particularly plasma samples) obtained from the subject is 24 units / L, more particularly 20 units / L, more particularly 16 units / L, more particularly 14 units / L, even most particularly 10 units / L, even more particularly 5 units / L. A specific threshold for bio-ADM levels is 15 pg / ml, more particularly 10 pg / ml, even more particularly 5 pg / ml.

[0157] In some implementations, a threshold is predetermined by measuring the ratio of peptide-Gly to peptide-amide (specifically ADM-Gly to bio-ADM) in the subject cohort and calculating, for example, the 75th percentile, more particularly the 90th percentile, or even more particularly the 95th percentile, to define whether a subject has a medical condition or is at risk of having a medical condition.

[0158] In other embodiments, the specific threshold for the ratio of ADM-Gly to bio-ADM in the sample obtained from the subject is 1, more particularly 1.5, more particularly 2, more particularly 2.5, more particularly 5, more particularly 7.5, or even more particularly 10.

[0159] One particular implementation relates to the use of PEGylated PAM in treating a disease characterized by peptide homeostasis disorder in a subject, particularly wherein the dose of PEGylated PAM administered is from 2.0 units / kg to 116.1 units / kg, particularly from 2.1 units / kg to 71.5 units / kg, more particularly from 2.2 units / kg to 54.1 units / kg, more particularly from 2.3 units / kg to 38.1 units / kg, more particularly from 2.4 units / kg to 24.1 units / kg, and even more particularly from 2.5 units / kg to 11.1 units / kg.

[0160] Obviously, this also relates to the use of unmodified PAM in subjects to treat diseases characterized by peptide homeostasis disorders and / or the use of modified PAM in subjects to treat diseases characterized by peptide homeostasis disorders. For clarification, specific embodiments also relate to the use of unmodified PAM in subjects to treat diseases characterized by peptide homeostasis disorders, particularly wherein the dosage of unmodified PAM administered is from 2.0 units / kg to 116.1 units / kg, particularly from 2.1 units / kg to 71.5 units / kg, more particularly from 2.2 units / kg to 54.1 units / kg, more particularly from 2.3 units / kg to 38.1 units / kg, more particularly from 2.4 units / kg to 24.1 units / kg, and even more particularly from 2.5 units / kg to 11.1 units / kg. For clarification, the specific implementation also relates to the use of modified PAM in treating diseases characterized by peptide homeostasis disorders in subjects, particularly wherein the dosage of the modified PAM is from 2.0 units / kg to 116.1 units / kg, particularly from 2.1 units / kg to 71.5 units / kg, more particularly from 2.2 units / kg to 54.1 units / kg, more particularly from 2.3 units / kg to 38.1 units / kg, more particularly from 2.4 units / kg to 24.1 units / kg, and even more particularly from 2.5 units / kg to 11.1 units / kg.

[0161] Furthermore, more specific embodiments relate to the use of PEGylated PAM in treating diseases characterized by peptide homeostasis disorders in subjects, particularly wherein the dosage of PEGylated PAM, more particularly PEG 5000 or PEG 10000, is from 2.0 units / kg to 116.1 units / kg, more particularly from 2.1 units / kg to 71.5 units / kg, more particularly from 2.2 units / kg to 54.1 units / kg, more particularly from 2.3 units / kg to 38.1 units / kg, more particularly from 2.4 units / kg to 24.1 units / kg, and even more particularly from 2.5 units / kg to 11.1 units / kg.

[0162] Furthermore, more specific embodiments relate to the use of XTEN-based PAM in treating diseases characterized by peptide homeostasis disorders in subjects, particularly wherein the dosage of XTEN-based PAM is from 2.0 units / kg to 116.1 units / kg, particularly from 2.1 units / kg to 71.5 units / kg, more particularly from 2.2 units / kg to 54.1 units / kg, more particularly from 2.3 units / kg to 38.1 units / kg, more particularly from 2.4 units / kg to 24.1 units / kg, and even more particularly from 2.5 units / kg to 11.1 units / kg.

[0163] In a particular implementation, the disease or condition characterized by peptide homeostasis disorder is selected from, but not limited to: Dementia, especially dementia with secondary causes (including intracranial lesions) selected from mild cognitive impairment (MCI), Alzheimer's disease, vascular dementia, mixed Alzheimer's disease and vascular dementia, Lewy body dementia, frontotemporal dementia, focal dementia (including progressive aphasia), subcortical dementia (including Parkinson's disease) and dementia syndromes. Cardiovascular diseases, especially those selected from atherosclerosis, hypertension, heart failure (including acute and acute compensated heart failure), atrial fibrillation, local ischemia of the cardiovascular system, ischemic brain injury, cardiogenic shock, stroke (including ischemic stroke and hemorrhagic stroke as well as transient ischemic attack), and myocardial infarction. Kidney diseases, especially those selected from nephrotoxic (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 or parasites, especially infectious bacteria, particularly diseases selected from SIRS, sepsis and septic shock; Metabolic diseases, especially those selected from type 1 diabetes, type 2 diabetes and metabolic syndrome.

[0164] In embodiments of the present invention, the pharmaceutical composition comprises a combination of modified or unmodified PAM and vitamin C.

[0165] In other embodiments, the pharmaceutical composition comprises a combination of modified or unmodified PAM and vitamin C, wherein the pharmaceutical composition may be used to treat and / or prevent type 2 diabetes.

[0166] In other embodiments, the pharmaceutical composition comprises a combination of modified or unmodified PAM and vitamin C, wherein the pharmaceutical composition may be used to treat and / or prevent IBD.

[0167] In other embodiments, the pharmaceutical composition comprises a combination of modified or unmodified PAM and vitamin C, wherein the pharmaceutical composition may be used to treat diseases or conditions selected from the group consisting of: cardiovascular, renal, inflammatory, infectious and metabolic diseases or conditions, dementia, cancer and other diseases or conditions related to peptide homeostasis disorders, wherein more particularly, the disease or condition is sepsis.

[0168] In other embodiments of the invention, combinations of modified or unmodified PAMs are used to treat and / or prevent cardiovascular, edematous, and / or inflammatory diseases, such as sepsis or septic shock, wherein the unmodified or modified PAMs are used in combination with vitamin C.

[0169] Other embodiments of the invention relate to the use of modified or unmodified PAM in treating diseases in subjects, wherein the modified or unmodified PAM is used in combination with vitamin C, and wherein the dosage of vitamin C is 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, and more preferably 10-1000 mg / kg.

[0170] One embodiment of the invention involves the use of modified or unmodified PAM in the treatment of a disease in a subject, wherein the modified or unmodified PAM is used in combination with vitamin C, and wherein... The preferred combination is 1.5-54 units / kg of PAM 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, and more preferably 10-1000 mg / kg of vitamin C. More preferably, a combination of 1.7-33 units / kg of PAM 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, and more preferably 10-1000 mg / kg of vitamin C. More preferably, a combination of 1.8-26 units / kg of PAM 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, and more preferably 10-1000 mg / kg of vitamin C. More preferably, a combination of 1.9-20.6 units / kg of PAM 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, and more preferably 10-1000 mg / kg of vitamin C. More preferably, a combination of 2-14 units / kg of PAM 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, and more preferably 10-1000 mg / kg of vitamin C. The administration dose can be a single bolus injection delivering the amount of the compound to be administered, or, taking into account the infusion rate, a continuous infusion of the compound over a specific time period to deliver the required amount of the compound. Therefore, the compound can be administered as a combination of injection and / or infusion solutions or as several parallel injection and / or infusion solutions, wherein one or all of the compounds are delivered as injection and / or infusion solutions, or one different compound is injected and another different compound is infused.

[0171] This includes, for example, the use of vitamin C as a standalone injection and / or infusion solution, and the use of modified or unmodified PAM as a standalone injection and / or infusion solution.

[0172] Furthermore, vitamin C and modified or unmodified PAM can be administered via the same or different routes of application if they are administered separately. This means, for example, vitamin C can be administered subcutaneously, while modified or unmodified PAM can be administered intravenously, with each administered separately.

[0173] It should be understood that both vitamin C and modified or unmodified PAM are administered as exogenous substances.

[0174] The compounds or combinations of the present invention are specifically administered intramuscularly, intraperitoneally, subcutaneously, or intravenously.

[0175] One embodiment of the present invention is a pharmaceutical combination or pharmaceutical composition in which vitamin C is used in combination with peptidyl glycine α-amidyl monooxygenase (PAM), wherein the PAM is modified by amino acid manipulation, preferably by fusion with albumin such as serum albumin or recombinant serum albumin, more preferably by non-covalent binding to serum albumin by linking to the conjugated fatty acid chain of PAM, more preferably by fusion with IgG Fc region or transferrin, preferably modified to be used as a PEG-based carrier prodrug, and most preferably by post-translational modification by linking a natural or synthetic polymer, wherein the natural or synthetic polymer used is HAP, preferably ELP, more preferably PAS, more preferably PSA, more preferably GLK, more preferably XTEN, and most preferably PEG.

[0176] Other embodiments of the invention relate to a method for sustainably increasing the levels of circulating amidated peptide hormones such as peptide-Gly by using its glycine extended precursor in combination with the activator PAM and, in one embodiment, with the cofactor vitamin C, and the use of this combination for treating and / or preventing acute and / or chronic diseases. The PAM enzyme is present in circulation and is capable of amidation in circulation.

[0177] This invention covers compounds or combinations of compounds suitable for the pharmaceutical industry. The compounds include not only the compound itself, but also pharmaceutically acceptable salts of the compound and solvents considered safe for pharmaceutical use. It is noteworthy that the pharmaceutically acceptable salts and solvates of the compounds are not limited to any specific type, but the salts or solvates listed above are preferred. When in salt or solvate form, the compounds can be used in a variety of pharmaceutical applications. The compounds should refer to a combination of PAM and vitamin C.

[0178] A specific embodiment of the present invention is as follows: 1. A modified peptidyl-glycine α-amidyl monooxygenase (PAM) having an increased in vivo half-life compared to unmodified PAM, wherein the enzymatic activity of the modified PAM is maintained compared to unmodified PAM.

[0179] 2. A method for increasing the in vivo half-life of PAM, wherein the method comprises modifying the PAM, wherein the in vivo half-life is increased compared to unmodified PAM, and wherein the enzymatic activity of the PAM is maintained.

[0180] In a particular embodiment of the invention, the in vivo half-life is increased in subjects. In a particular embodiment, the in vivo half-life is increased in subjects who have received at least one administration of the modified PAM.

[0181] The preservation of PAM's enzymatic activity means that the modified PAM possesses the PAM enzymatic activity as further described herein. In certain embodiments, the enzymatic activity of the PAM is its in vitro or in vivo, particularly in vivo, enzymatic activity. In particular, this relates to the amidation activity of the PAM as described herein.

[0182] 3. The modified PAM according to Embodiment 1 or the method according to Embodiment 2, wherein the PAM is a protein comprising a polypeptide sequence selected from SEQ ID No. 1 to 10 or having at least 85%, particularly at least 90%, more particularly at least 95%, or even more particularly at least 99% sequence homology therewith, alternatively wherein sequence homology therewith means 85% to 100%, preferably 90% to 100%, or more preferably 95% to 100%, or most preferably 99% to 100% sequence homology.

[0183] 4. The modified PAM or method according to any of the foregoing embodiments, wherein the enzymatic activity of the PAM is the α-amidation activity of the PAM.

[0184] 5. The modified PAM or method according to any of the foregoing embodiments, wherein the PAM is modified in the following manner

[0185] a. Connecting one or more natural or synthetic polymer units, wherein the polymer is selected from the group consisting of: i. PEG, particularly in the range of average molecular weight from 0.2 to 100 kDa, particularly from 1 to 90 kDa, more particularly from 2 to 80 kDa, more particularly from 3 to 70 kDa, more particularly from 4 to 60 kDa, and even more particularly from 5 to 50 kDa; ii. XTEN; iii. Peptide polymers composed of repeating PAS (amino acids proline, alanine, and serine) units, particularly containing 100-200, particularly 120-180, more particularly 130-170, and even more particularly about 150 PAS repeating units; iv. Elastin-like polypeptides composed of repeating units of valine-proline-glycine-x-glycine, wherein x is any amino acid other than proline, and particularly composed of 150, 200, 250, 300, 400 or 500 to 550, 600, 750, 850, 950 or 1000 amino acids. v. HAP polypeptide, with the sequence (Gly4Ser)n, where n is 100 to 200; vi. Gelatin-like fusion protein with the sequence (Gly-XY)n, where X and Y are any amino acids except cysteine, and where n = 60 to 1500; vii. Polysaccharides, especially those selected from the following group: 1. Glucan; 2. Hydroxyethyl polysaccharides; 3. Proheparin; 4. Hyaluronic acid; viii. Polysialic acid; b. Conjugated to serum proteins such as albumin or immunoglobulins or partial conjugations of immunoglobulins; c. Site-directed mutagenesis by inserting, deleting, and / or exchanging one or more amino acids within the amino acid sequence of PAM.

[0186] 6. The modified PAM or method according to any of the foregoing embodiments, wherein the in vivo half-life of the modified PAM is increased by at least 2 times, particularly at least 5 times, more particularly at least 10 times, more particularly at least 20 times, more particularly at least 50 times, more particularly at least 100 times, more particularly at least 150 times, or alternatively 1.05 to 2 times, or 1.05 to 5 times, or 1.05 to 10 times, or 1.05 to 20 times, or 1.05 to 50 times, or 1.05 to 100 times, or 1.05 to 125 times, or 1.05 to 150 times.

[0187] 7. The modified PAM or method according to any of the foregoing embodiments, wherein the PAM level in a sample obtained from a subject receiving at least one dose of the modified PAM is increased compared to a pre-drug sample obtained from the subject before receiving the at least one dose of the modified PAM, wherein the sample is 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 after the last dose of the modified PAM is administered to the subject. The sample was obtained from the subject at 120 hours, more particularly 144 hours, or more particularly 168 hours after the last dose of the modified PAM was administered, or alternatively, the sample was obtained from the subject at 2 hours, more 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, or more particularly 2 to 168 hours after the last dose of the modified PAM was administered.

[0188] In a particular implementation, the increase in PAM levels involves an increase in total PAM levels, including modified PAM and unmodified PAM (wherein the unmodified PAM is naturally present in the subject).

[0189] 8. The modified PAM or method according to any of the foregoing embodiments, wherein the increase in PAM level and / or the increase in PAM half-life is determined by measuring α-amidation activity and / or amidated peptide hormone levels and / or by measuring PAM concentration in a sample obtained from a subject who has received at least one dose of the modified PAM.

[0190] 9. The modified PAM or method according to any of the foregoing embodiments, wherein the in vivo half-life of the modified 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, or 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.

[0191] 10. The modified PAM or method according to any of the foregoing embodiments, wherein the in vivo half-life of the 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, or 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.

[0192] 11. The modified PAM or method according to any of the foregoing embodiments, wherein the in vivo half-life is the half-life level in the subject's blood circulation.

[0193] 12. The modified PAM or method according to any of the foregoing embodiments, wherein at least 50%, particularly at least 75%, particularly at least 85%, particularly at least 95%, particularly about 100% of the enzyme activity is maintained compared to unmodified PAM, alternatively wherein maintaining enzyme activity means that the enzyme activity is 50% to 100%, preferably 75% to 100%, more preferably 85% to 100%, more preferably 95% to 100%, or most preferably 100% compared to unmodified PAM.

[0194] 13. The modified PAM or method according to any of the foregoing embodiments, wherein the modified PAM, when administered to a subject, increases the bioavailability of one or more amidated peptides, particularly one or more peptide hormones, in the subject.

[0195] 14. A pharmaceutical composition comprising modified PAM according to any one of embodiments 1 to 13.

[0196] 15. The pharmaceutical composition according to embodiment 14, wherein the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients, such as surfactants, emulsifiers or carriers.

[0197] 16. The pharmaceutical composition according to embodiment 14 or 15, wherein the pharmaceutical composition is a ready-to-use formulation.

[0198] 17. The pharmaceutical composition according to any one of embodiments 14 to 16, wherein the pharmaceutical composition is a freeze-dried composition.

[0199] In some embodiments, the pharmaceutical composition according to the invention comprises one or more items selected from the packaging instructions, instructions for use to the subject and / or the physician treating the subject, and product information leaflets. These items may in particular include instructions regarding the administration of the pharmaceutical composition to the subject, such as instructions regarding dosage, indications, and route of administration.

[0200] 18. The modified PAM according to any one of embodiments 1 to 13 or the pharmaceutical composition according to any one of embodiments 14 to 17, used as a medicament.

[0201] Some embodiments of the present invention relate to the use of the modified PAM or pharmaceutical composition according to the present invention in the manufacture of a pharmaceutical product.

[0202] Some embodiments of the present invention relate to the use of the modified PAM or pharmaceutical composition according to the present invention for the treatment of medical conditions.

[0203] Some embodiments of the present invention relate to a method of treating a subject in need, the method comprising administering to the subject a therapeutically effective amount of a modified PAM or pharmaceutical composition according to the present invention.

[0204] This invention relates to specific embodiments of modified PAM or pharmaceutical compositions used as medicines, specifically embodiments of modified PAM or pharmaceutical compositions used to treat or prevent certain medical conditions or to reduce the risk of a subject contracting a medical condition, with reference to the above-described uses for manufacturing medicines, uses for treating medical conditions, and / or methods of treatment.

[0205] 19. A modified PAM according to any one of embodiments 1 to 13 or a pharmaceutical composition according to any one of embodiments 14 to 17, for reducing the risk of a subject contracting a medical condition, and / or for reducing the incidence of a disease or symptom in the subject, and / or for reducing the severity of a disease or symptom in the subject.

[0206] 20. A modified PAM according to any one of embodiments 1 to 13 or a pharmaceutical composition according to any one of embodiments 14 to 17, for achieving an increase in PAM level and / or α-amidation activity level in the blood circulation of a subject, particularly an increase of at least 2 times, particularly at least 3 times, particularly at least 5 times, particularly at least 9 times, particularly at least 10 times, 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.

[0207] In this invention, subjects treated with the modified PAM or pharmaceutical composition according to the invention are particularly those who require increased PAM levels and / or increased bio-ADM levels and / or increased levels of another mature peptide, wherein the other peptide is a peptide present in vivo in a precursor form containing a C-terminal glycine and convertible into a mature peptide form having a C-terminal amide.

[0208] 21. A modified PAM according to any one of embodiments 1 to 13 or a pharmaceutical composition according to any one of embodiments 14 to 17, for the treatment of diseases or conditions selected from the group consisting of: cardiovascular, renal, inflammatory, infectious and metabolic diseases or conditions, dementia, cancer and other diseases or conditions related to peptide homeostasis disorders.

[0209] 22. A modified PAM or pharmaceutical composition for treating a disease or condition according to embodiment 21, wherein the disease or condition is sepsis.

[0210] In a particular embodiment of the invention, the modified PAM or the pharmaceutical composition is formulated for administration via, or via, oral, epidermal, subcutaneous, intradermal, sublingual, intramuscular, intra-arterial, intravenous, central nervous system (CNS, intracerebral, intraventricular, intrathecal) or intraperitoneal administration, particularly via the epidermal, subcutaneous, intradermal, intramuscular or intraperitoneal route, more particularly via the subcutaneous, intramuscular or intraperitoneal route; “formulated for administration (e.g., oral)” is used mutatis mutandis with “formulated for (e.g., oral) administration”.

[0211] In a preferred embodiment of the invention, the pharmaceutical composition is formulated for administration via, or via, the following routes: epidermal, subcutaneous, intradermal, intramuscular, intraarterial, intravenous, via the central nervous system (CNS, intracerebral, intraventricular, intrathecal), or via the peritoneal cavity, particularly via the epidermal, subcutaneous, intradermal, intramuscular, or peritoneal cavity, more particularly via the subcutaneous, intramuscular, or peritoneal cavity; “formulated for administration (e.g., oral)” is used mutatis mutandis with “formulated for (e.g., oral) administration”.

[0212] In the most preferred embodiment of the invention, the pharmaceutical composition is formulated for administration via subcutaneous, intradermal, intramuscular, intra-arterial, or intravenous routes, or via intraperitoneal administration.

[0213] According to the present invention, the administration dose may be a single bolus delivering the amount of the compound to be administered, or, taking into account the infusion rate, a continuous infusion of the compound delivering the required amount of the compound over a specific time period.

[0214] Other embodiments of the present invention also relate to: 1. A modified peptidyl-glycine α-amidyl monooxygenase (PAM) having an increased in vivo half-life compared to unmodified PAM, wherein the enzymatic activity of the modified PAM is maintained compared to unmodified PAM.

[0215] 2. A method for increasing the in vivo half-life of PAM, wherein the method comprises modifying the PAM, wherein the in vivo half-life is increased compared to unmodified PAM, and wherein the enzymatic activity of the PAM is maintained.

[0216] 3. The modified PAM according to Embodiment 1 or the method according to Embodiment 2, wherein the PAM is a protein comprising a polypeptide sequence selected from SEQ ID No. 7 to 18 or having at least 85%, particularly at least 90%, more particularly at least 95%, or even more particularly at least 99% sequence homology with it.

[0217] 4. The modified PAM according to Embodiment 1 or the method according to Embodiment 2, wherein the PAM is a protein comprising a polypeptide sequence selected from SEQ ID No. 7 to 16 or having at least 85%, particularly at least 90%, more particularly at least 95%, or even more particularly at least 99% sequence homology with it.

[0218] 5. The modified PAM or method according to any of the foregoing embodiments, wherein the enzymatic activity of the PAM is the α-amidation activity of the PAM.

[0219] 6. The modified PAM or method according to any of the foregoing embodiments, wherein the PAM is modified in the following manner

[0220] a. Connecting one or more natural or synthetic polymer units, wherein the polymer is selected from the group consisting of: i. PEG, particularly in the range of average molecular weight from 0.2 to 100 kDa, particularly from 1 to 90 kDa, more particularly from 2 to 80 kDa, more particularly from 3 to 70 kDa, more particularly from 4 to 60 kDa, and even more particularly from 5 to 50 kDa; ii. XTEN; iii. Peptide polymers composed of repeating PAS (amino acids proline, alanine, and serine) units, particularly containing 100-200, particularly 120-180, more particularly 130-170, and even more particularly about 150 PAS repeating units; iv. Elastin-like polypeptides composed of repeating units of valine-proline-glycine-x-glycine, wherein x is any amino acid other than proline, and particularly composed of 150, 200, 250, 300, 400 or 500 to 550, 600, 750, 850, 950 or 1000 amino acids. v. HAP polypeptide, with the sequence (Gly4Ser)n, where n is 100 to 200; vi. Gelatin-like fusion protein with the sequence (Gly-XY)n, where X and Y are any amino acids except cysteine, and where n = 60 to 1500; vii. Polysaccharides, especially those selected from the following group: 1. Glucan; 2. Hydroxyethyl polysaccharides; 3. Proheparin; 4. Hyaluronic acid; viii. Polysialic acid; b. Conjugated to serum proteins such as albumin or immunoglobulins or partial conjugations of immunoglobulins; c. Site-directed mutagenesis by inserting, deleting, and / or exchanging one or more amino acids within the amino acid sequence of PAM.

[0221] 7. The modified PAM or method according to any of the foregoing embodiments, wherein the in vivo half-life of the modified PAM is increased by at least 2 times, particularly at least 5 times, more particularly at least 10 times, more particularly at least 20 times, more particularly at least 50 times, more particularly at least 100 times, more particularly at least 150 times, more particularly at least 250 times, more particularly at least 350 times, and / or

[0222] Compared to unmodified PAM, the modified PAM exhibits an in vivo half-life 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

[0223] The in vivo half-life of the 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, and more particularly at least 60 hours.

[0224] 8. The modified PAM or method according to any of the foregoing embodiments, wherein the PAM level in a sample obtained from a subject who has received at least one dose of the modified PAM is increased compared to a pre-drug sample obtained from the subject before receiving the at least one dose of the modified PAM, wherein the sample is obtained from the 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 the last dose of the modified PAM is administered.

[0225] 9. The modified PAM or method according to any of the foregoing embodiments, wherein at least 50%, particularly at least 75%, particularly at least 85%, particularly at least 95%, particularly about 100% of the enzyme activity is maintained compared to the unmodified PAM.

[0226] 10. The modified PAM or method according to any of the foregoing embodiments, wherein the modified PAM, when administered to a subject, increases the bioavailability of one or more amidated peptides, particularly one or more peptide hormones, in the subject.

[0227] 11. A pharmaceutical composition comprising modified PAM according to any one of embodiments 1 to 10.

[0228] 12. The pharmaceutical composition according to embodiment 11, wherein the pharmaceutical composition is an ready-to-use formulation.

[0229] 13. The pharmaceutical composition according to any one of embodiments 11 to 12, wherein the pharmaceutical composition is a freeze-dried composition.

[0230] 14. A modified PAM according to any one of embodiments 1 to 10 or a pharmaceutical composition according to any one of embodiments 11 to 13, for achieving an increase in PAM level and / or α-amidation activity level in the blood circulation of a subject, particularly an increase of at least 2 times, particularly at least 3 times, particularly at least 5 times, particularly at least 9 times, particularly at least 10 times, 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.

[0231] 15. The modified PAM according to any one of embodiments 1 to 10 or the pharmaceutical composition according to any one of embodiments 11 to 13, used as a medicine, particularly for treating diseases or conditions selected from the group consisting of: cardiovascular, renal, inflammatory, infectious and metabolic diseases or conditions, dementia, cancer and other diseases or conditions associated with peptide homeostasis disorders, wherein more particularly, said diseases or conditions are sepsis.

[0232] 16. A modified PAM according to any one of embodiments 1 to 10 or a pharmaceutical composition according to any one of embodiments 11 to 13, for reducing the risk of a subject contracting a medical condition, and / or for reducing the incidence of a disease or symptom in the subject, and / or for reducing the severity of a disease or symptom in the subject.

[0233] 17. A modified PAM according to any one of embodiments 1 to 10 or a pharmaceutical composition according to any one of embodiments 11 to 13, for use in treating or preventing a disease or medical condition in a subject, wherein the disease or medical condition is associated with endothelial barrier dysfunction and / or blood-brain barrier dysfunction.

[0234] 18. The modified PAM for treating or preventing a disease in a subject according to embodiment 17, wherein the disease or medical condition is selected from the group consisting of: cardiovascular, renal, inflammatory, infectious and metabolic diseases or conditions, dementia, cancer and other diseases or medical conditions associated with peptide homeostasis disorders, wherein more particularly, the disease or condition is sepsis. Detailed Implementation

[0235] Example

[0236] Example 1 - PAM activity in sepsis and septic shock

[0237] PAM activity (AMA) in Li-heparinized plasma was measured in a randomized cohort of 199 individuals in the prospective, observational, multinational adrenaline-medulloin and sepsis and septic shock 1 prognostic study (AdrenOSS-1). A cohort including 98 self-reported healthy individuals served as a control group. AMA was determined using the method described in Example 3.

[0238] Circulating AMA levels were measured in Li-heparinized plasma upon admission to the intensive care unit (ICU). Median AMA levels in sepsis and septic shock were significantly elevated compared to 18.4 units / L [13.5–21.9 units / L] in healthy individuals, at 23.5 units / L [IQD: 17.7–32.7 units / L] and 22.4 units / L [IQD: 18.2–38.9 units / L], respectively. Figure 4 A). Patients with AMA levels higher than 35.1 units / L upon ICU admission had a survival rate of approximately 15% in sepsis and septic shock, while higher amidation activity resulted in a mortality rate of approximately 40%. Figure 4 B).

[0239] Example 2 - Determination of ADM-Gly concentration in sepsis and septic shock

[0240] In a randomized cohort of 199 individuals from the AdrenOSS-1 study, ADM-Gly concentrations in Li-heparinized plasma were measured. A cohort of 100 self-reported healthy individuals served as a control group. ADM-Gly concentrations were measured using a one-step high-throughput microtiter plate-based chemiluminescent immunoassay, where the solid phase and tracer antibody specifically targeted the central region (amino acids 21–42) and C-terminal region (amino acids 42–52) of ADM-Gly, respectively.

[0241] Circulating ADM-Gly levels were measured in Li-heparinized plasma upon ICU admission. Median ADM-Gly concentrations were significantly higher in patients with sepsis (121.5 pg / mL [IQD: 44.4-284.1 pg / mL]) and septic shock (419.3 pg / mL [IQD: 148.8-1106 pg / mL]) compared to self-reported healthy individuals (17.5 pg / mL [IQD: 11.5-27.2 pg / mL]). Figure 5 A). Elevated ADM-Gly levels were found to be associated with higher mortality rates from sepsis and septic shock.

[0242] Patients with ADM-Gly values ​​below 730 pg / mL upon ICU admission have a better survival prognosis; the mortality rate from sepsis and septic shock is approximately 15%. Figure 5 B). In contrast, the 28-day mortality rate for patients with ADM-Gly values ​​higher than 730 pg / mL was approximately 50%. Figure 5 B).

[0243] Example 3 - PAM Activity Assay

[0244] Human serum or Li-heparinized plasma from self-reported healthy volunteers was used as the source of human natural PAM. Each sample (20 μl) was diluted twice in 100 mM Tris-HCl buffer, in duplicate. Amidation was initiated by adding 160 μL of PAM reaction buffer (100 mM Tris-HCl pH 7.5, 6.25 μM CuSO4, 2.5 mM L-ascorbate, 125 μg / mL catalase, 62.5 μM amoxicillin, 250 μM leucine, 36 ng / mL synthetic ADM-Gly, and 375 μg / mL NT-ADM antibody). Then, 100 μl of each of the two individual reactions was combined and transferred to 20 μl of 200 mM EDTA solution to terminate the amidation and generate a t=0 min reaction time point. The mixture was then incubated at 37°C for 40 min, after which any unterminated reaction was terminated by adding 10 μl of 200 mM EDTA solution. To determine PAM activity, bio-ADM as a reaction product in each sample was quantified using the sphingotest® bio-ADM immunoassay (Weber et al., 2017). The amidation assay was calibrated using a 6-point calibration curve generated with known-activity human recombinant PAM. Samples and calibrators were treated in the same manner. The relative light units (RLU t40 min–t0 min) of each sample, as determined by the sphingotest bio-ADM immunoassay, were fitted to the RLU of the calibrator (t40 min–t0 min) to determine the PAM activity (AMA) in the sample. AMA is expressed in units, where 1 AMA unit is defined as 1 μg bio-ADM formed per hour. Units / L describe AMA as 1 μg bio-ADM formed per L of sample per hour.

[0245] Typical PAM calibration curves are as follows Figure 6 As shown. Figure 7The distribution of AMA in Li-heparin samples from n=120 self-reported healthy volunteers is shown. The median [IQR] of Li-heparin AMA was 18.4 units / L [13.5–21.9]. The 10th and 90th percentiles were 10.5 and 24.2 units / L, respectively. The 2nd, 97th, and 99th percentiles were 8.1, 31.6, and 40.8 units / L, respectively. Furthermore, matched serum samples from n=20 subjects were measured and showed a highly significant correlation (r=0.89; p<0.0001), but serum AMA values ​​were approximately 40% lower than those in Li-heparin.

[0246] Example 4 - Generation of Recombinant PAM

[0247] Variant A: PAM cDNA was synthesized according to Uniprotocol accession number P19021, encoding amino acids 21-834 of the PAM protein, including codon optimization for expression in mammalian cells. The signal sequence of PAM was replaced with the human serum albumin signal sequence (MKWVTFISLLFLFSSAYSFR [SEQ ID No. 9]). A hexahistine tag was added to the C-terminus of PAM and linked to PAM via a GS adapter. The sequence of recombinant PAM (amino acids 21-834 of PAM without the signal sequence and hexahistine tag) is shown in SEQ ID No. 10. The cDNA was cloned into an expression vector (plasmid DNA) using 5'-NotI and 3'HindIII restriction sites. The expression vector containing the cDNA for PAM expression was replicated in E. coli and prepared as a low-endotoxin formulation by E. coli.

[0248] HEK-INV cells were transfected with the expression vector using the INVect transfection reagent in serum-free suspension culture. Transfection rate was controlled by co-transfection with an expression vector containing GFP (green fluorescent protein). Cells were cultured at 37°C and 5% CO2 in the presence of valproic acid and penicillin-streptomycin. Cells were harvested by centrifugation (>2000 g, 30–45 min, 2–8°C) when viability reached <60%. Cell culture supernatant (CCS) was washed five times with 100 mM Tris / HCl pH 8.0 via tangential flow filtration (TFF, 30 kDa cutoff).

[0249] Purification of recombinant PAM involved applying buffer-exchanged CCS to a Q-agarose fast flow resin (GE Healthcare) and eluting with a NaCl gradient (up to 2 M). Fractions containing amidation activity were pooled and applied to a Superdex 200 pg (GE Healthcare) size exclusion column containing 100 mM Tris / HCl, 200 mM NaCl, pH 8.0 elution buffer. Fractions containing amidation activity were pooled and dialyzed against 100 mM Tris / HCl, 200 mM NaCl, pH 8.0, followed by sterile filtration (0.2 μm). Endotoxin load was determined using a Charles River PTS Endosafe system and was less than 5 EU / mL.

[0250] Variant B: A second construct of full-length PAM for immunization was purchased from SinoBiological and contains residues 31-818 of human PAM (UniProtKB: P19021-1, SEQ ID No.1) and is tagged with a decahistidine tag at the C-terminus.

[0251] Example 5: PAM modification: PEGylation with PEG-5000

[0252] 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 PEGylating recombinant PAM using PEG-5000.

[0253] Procedure: Dissolve 10 mg of lyophilized PAM in 5 mL of pH 10 phosphate-buffered saline (PBS). Next, add 1 M NaOH to adjust the pH to 8.5. Prepare a 20 mM PEG-5000 solution by dissolving 70.5 mg of PEG-5000 in 705 μL of 20% DMSO. Add this solution to the PAM solution at approximately a 90-120 molar excess compared to PAM, or at a 2.2 molar excess compared to the lysine residues present in the PAM construct. Incubate the PAM-PEG mixture at 4 °C with continuous shaking for 120 min. After 90 min, add 500 μL of 1 M Tris solution to the mixture to inactivate any unreacted PEG, and incubate for another 30 min. To separate PEG-PAM from free PEG, fractionate the mixture on a Superdex 200 column (ÄKTAStart) using PBS as the run buffer. A protein peak was detected at 280 nm, and fractions corresponding to molecular weights from 160 to 90 kDa were collected and combined. The combined fractions were aseptically filtered using a 0.2 μm filter and aliquoted under aseptic conditions for further use in in vivo experiments.

[0254] Results: Gel filtration analysis showed a broad peak between 200 kDa and approximately 80 kDa, indicating different degrees of PAM PEGylation. Figure 8 SDS-PAGE results further confirmed this finding, showing PEG-PAM tailing, indicating varying degrees of PEGylation. The highest UV absorption peak at 280 nm corresponds to a molecular weight of 130 kDa, indicating successful PEGylation and molecular weight increase. Furthermore, the absence of high-molecular-weight compounds in the UV spectrum suggests that the current PEGylation conditions do not induce protein aggregation. Additionally, the enzymatic activity of PEG-PAM is comparable to that of untreated PAM, indicating that the PEGylation protocol does not interfere with enzyme activity.

[0255] For this purpose, the protein concentrations of PEGylated (PEG-PAM) and unmodified recombinant PAM were determined using a dioctanine protein assay kit (Micro BCA™ Protein Assay Kit, ThermoFisher Scientific, catalog number 23235). To evaluate their respective activities, three samples of PEGylated PAM and unmodified 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.

[0256] like Figure 9As shown, the results indicate that the activity of PEGylated PAM was slightly reduced compared to unmodified PAM. Specifically, at a PAM concentration of 2 μg / mL, PEGylated PAM exhibited a 14.2% decrease in activity. Similarly, at a concentration of 1 μg / mL, PEGylated PAM showed a 6.1% decrease in activity. Finally, for the sample at a concentration of 0.5 μg / mL, PEGylated PAM showed a 9.3% decrease in activity.

[0257] Example 6: Single intravenous injection of untreated and PEGylated PAM

[0258] The in vivo half-life of PEGylated PAM (PEG-PAM) and unmodified PAM (Example 4, variant B) in rat blood circulation was compared. A single intravenous injection of 14.4 units per kg of animal body weight (approximately corresponding to 6 units / rat) was administered. Li-heparin plasma was collected at 20, 40, 60, 80, 120, 180, 240, and 300 minutes after intravenous injection, and PAM amidation activity was measured using the method described in Example 3. Plasma samples were collected from three rats at each time point.

[0259] Results: Even 300 minutes after injection, PEG-PAM still exhibited high amidation activity. Figure 10 In contrast, unmodified PAM lost 75% of its original activity 180 minutes after injection. This finding is consistent with the previously reported half-life (47 minutes) of unmodified PAM in circulation (Kaufmann et al., 2021). Surprisingly, the half-life of PEG-PAM was found to be greater than 300 minutes, which equates to an increase of more than six times compared to unmodified PAM.

[0260] Example 7: PEG-PAM administration route and half-life in cycling

[0261] The study investigated the enhancement of plasma amidation activity (AMA) in rats following administration of PEG-PAM via subcutaneous (sc), intramuscular (im), and intraperitoneal (ip) routes. A control group using untreated PAM (Example 4, variant B) was also included. The doses used were 14.4 units of PEG-PAM or unmodified PAM per kg of animal body weight, equivalent to approximately 6 units / rat. Li-heparinized plasma was collected at different time points, including 15, 30, 60, 120, 4, 8, 12, 24, and 72 hours after injection, and AMA was measured as described in Example 3. Plasma samples were taken from three rats at each time point.

[0262] The half-life of modified (PEGylated) and unmodified PAM is described as the time required for its activity to halve after reaching a plateau phase following bolus administration (Table 1).

[0263] Table 1: Circulating half-life of unmodified and modified PAM (PEGylated) after intravenous, intraperitoneal, subcutaneous and intramuscular bolus administration.

[0264]

[0265] Figure 11 The study showed a significant increase in plasma AMA 2 hours after subcutaneous injection of PEG-PAM, with activity 40% higher than pre-injection levels. At 4 hours, plasma AMA was 110% higher than baseline, reaching its maximum at 24 hours post-injection, with an increase of 984% compared to baseline activity. Even 3 days post-injection, AMA remained elevated, with levels 585% higher than pre-injection levels. In contrast, unmodified PAM showed a slight increase in activity of 46% at 4 hours post-injection, returning to baseline levels after 8 hours.

[0266] Similarly, an increase in plasma AMA was observed when PEG-PAM was administered intraperitoneally. Figure 12 Fifteen minutes after intraperitoneal injection, plasma AMA increased by 40%, reaching its peak activity eight hours after injection, representing a 1900% increase compared to baseline amidation activity. This increase in AMA remained constant over the next 16 hours, and even three days after injection, AMA was still elevated by 710%. Intraperitoneal injection of unmodified PAM showed a small, sustained increase in plasma AMA 15 minutes after injection, reaching its peak activity four hours after injection, showing a 55% increase compared to baseline activity, and returning to baseline activity eight hours after injection.

[0267] In addition, intramuscular administration of PEG-PAM also led to an increase in plasma AMA ( Figure 13 An increase in AMA of 153% was observed 15 minutes after intramuscular injection, peaking at 1680% 4 hours later. This increase in AMA remained constant over the next 20 hours, and even 3 days after injection, AMA was still elevated by 935%. Intramuscular injection of unmodified PAM showed a 185% increase in plasma AMA compared to baseline activity 2 hours after intramuscular injection. AMA returned to baseline activity 12 hours after injection.

[0268] In summary, these results indicate that all three administration routes are excellent options for increasing plasma PAM amidation activity.

[0269] Example 8: Protective effect of PEG-PAM on the blood-brain barrier

[0270] Sepsis is the result of an acute systemic immune response to various harmful injuries, particularly bacterial infections. This response activates numerous host mediator systems, including cytokines, leukocytes, complement, and the coagulation homeostasis network, each of which can contribute to the pathological sequelae of sepsis. Subsequently, the immune response triggers vascular endothelial cell damage, disrupting tight junction proteins; the blood-brain barrier (BBB) ​​is thus breached, allowing and facilitating the entry of peripheral immune cells into the brain, thereby triggering or exacerbating glial cell activation and neuroinflammation.

[0271] Male C57Bl / 6N mice aged 12–15 weeks (Charles River, Germany) were used. All procedures were performed according to the guidelines of the German Society for Animal Science (Gesellschaft für Versuchstierkunde; GV-SOLAS).

[0272] Mice were anesthetized with isoflurane (induction concentration 3%, maintenance concentration 1.5%, oxygen flow rate 3 L / min). A 1 cm abdominal incision was made along the ventral midline, and the cecum was ligated distal to the ileocecal valve with 4-0 silk sutures and punctured with a 24-gauge needle. A 2-3 mm fecal droplet was expelled. The incision was closed with 4-0 surgical sutures. Immediately postoperatively, mice were resuscitated intraperitoneally with 500 μl of preheated saline. Animals in the sham-operated group underwent the same surgery except for CLP. For preoperative treatment, 0.1 mg / kg buprenorphine (subcutaneously) + 100 mg / kg aminopyrine were administered parenterally. For postoperative analgesia, 0.1 mg / kg buprenorphine was administered subcutaneously twice daily (morning and evening). Animals were followed up for 24 hours. Each group consisted of n=12 mice (1 treatment group, 1 placebo group, 1 treatment sham-operated group, and 1 placebo sham-operated group).

[0273] The protective effect of PEGylated PAM on blood-brain barrier integrity was investigated. 1×PBS was used in the placebo group. Placebo or PEG-PAM was administered intraperitoneally (ip) on day -1 prior to CLP. The dose of PEG-PAM was 14.4 units / kg.

[0274] At the end of the observation period (24 hours), the animals were intravenously injected with Evans blue dye solution (4 mg / kg, dissolved in PBS at 1 mg / ml). Blue discoloration of the limbs indicated that the dye had entered the bloodstream.

[0275] In the presence of a functional brain blood flow barrier (BBB), the dye does not cross the BBB. In cases of BBB damage, the dye can enter the brain tissue. Mice were sacrificed after 30 minutes, and to remove the dye from the blood vessels, they were perfused systemically with PBS through the left ventricle until the fluid in the right atrium was colorless. The brain was removed, photographed, weighed, and homogenized in 1×PBS. The homogenate was centrifuged at 10,000×g for 20 minutes. The absorbance of the supernatant at 620 nm was measured using a known concentration of EB dye as a standard to quantify the presence of EB dye.

[0276] Figure 14 This study demonstrates that PEG-PAM pretreatment can prevent blood-brain barrier disruption induced by systemic inflammation caused by sepsis in rodent models with impaired vascular integrity. The degree of blood-brain barrier leakage, expressed as the concentration of Evans blue dye per 1 g of brain homogenate (μg / mL), was comparable in the sham-operated group treated with PEG-PAM or PBS, and in the CLP-induced group treated with PAM (approximately 4 μg EB / g brain homogenate). In contrast, the CLP-induced group treated with PBS showed a higher concentration, approximately 13 μg EB / g homogenate.

[0277] The data obtained clearly demonstrate that PEG-PAM treatment has a preventive effect on blood-brain barrier disruption, and therefore can be used to prevent or treat blood-brain barrier disruption in a range of diseases, including but not limited to MCI, dementia, Alzheimer's disease, stroke, sepsis, and septic shock.

[0278] Example 9: PAM modification: PEGylation was performed using PEG-10000, PEG-5000 via cysteine ​​and XTEN peptide. change

[0279] The following modification scheme provides a reproducible and effective method for modifying recombinant PAM (Example 4) with Mal-PEG-5000 (5 kDa PEG), NHS-PEG-10000 (10 kDa PEG) and / or XTEN peptide (SEQ ID No. 19).

[0280] Protocol: Use 1.5 mL of pH 10 phosphate-buffered saline (PBS) containing 1.5 mg of recombinant PAM. Add 1 M NaOH to adjust the pH to 8.5, and divide the preparation into three 500 μl portions.

[0281] When PEGylation was performed using NHS-PEG-10000 (11153 Da, Iris Biotech GmbH, Germany), a 10 mM NHS-PEG-10000 solution was prepared by dissolving 62 mg of PEG-10000 in 556 μL of 20% DMSO. This solution was added to the PAM solution in an excess of approximately 90–120 moles compared to PAM, or in an excess of 2.2 moles compared to the lysine residues present in the PAM construct. The PAM-PEG mixture was incubated at 4 °C with continuous shaking for 120 min. After 120 min, 50 μL of 2 M Tris solution was added to the mixture to inactivate any unreacted NHS-PEG, and incubation was continued for another 30 min. To separate PEG-PAM from free PEG, the mixture was fractionated on a Superdex 200 column (ÄKTA Start) using PBS as the run buffer. A protein peak was detected at 280 nm, and fractions corresponding to molecular weights from 160 to 90 kDa were collected and combined. The combined fractions were aseptically filtered using a 0.2 μm filter and aliquoted under aseptic conditions.

[0282] For PEGylation with MAL-PEG-5000 (4908 Da, Iris Biotech GmbH, Germany), a 1 MDTT solution was prepared, and 5 μl of this solution was added to a second 500 μl PAM fraction. After incubation at ambient temperature for 1 hour, the PAM protein was desalted using a G5 desalting column (EMP Biotech, Germany) to a final volume of 1 mL. A 20 mM MAL-PEG-5000 solution was prepared by dissolving 65 mg of MAL-PEG in 662.2 μl of 20% DMSO. This solution was added to the PAM solution in approximately a 90-120 molar excess compared to the PAM. After incubation at ambient temperature for 120 minutes with continuous shaking and in the dark, PEG-PAM was separated from free PEG by fractionation on a Superdex 200 column (ÄKTA Start) using PBS as the running buffer. A protein peak was detected at 280 nm, and fractions corresponding to molecular weights from 160 to 90 kDa were collected and combined. The combined fractions were aseptically filtered using a 0.2 μm filter and aliquoted under aseptic conditions.

[0283] When XTENation is performed using an XTEN peptide (3623.7 Da, custom-synthesized by Peptides & Elephants GmbH, Germany) having the following amino acid sequence H-SESATPESGPGSEPATSGSETPGTSESATPESC-OH (SEQ ID No. 19), a third 500 μl PAM is used.

[0284] In the first step, PAM was coupled to the linker MAL-β-Ala-Osu (maleimide butyryloxysuccinimide ester, Iris Biotech GmbH, Germany) by adding 15 μl of 10 mM MAL-β-Ala-Osu (in 20% DMSO) solution to the PAM protein, thereby coupling the linker to the free lysine residues of the PAM protein via its N-succinimide ester. The reaction was incubated at 4°C with continuous shaking for 120 min. After 120 min, 50 μL of 2 M Tris solution was added to the mixture to inactivate any unreacted linkers, and incubation was continued for another 30 min. Then, the PAM protein was desalted to PBS pH 10 using a G5 desalting column (EMP Biotech, Germany) to remove unreacted linkers, with a final volume of 1 mL. A 19.3 mM XTEN-peptide solution was prepared in PBS pH 10, and a 100-fold molar excess of 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 incubation at ambient temperature with continuous shaking and in the dark for 120 min, XTENated PAM (X-PAM) and free XTEN were separated by fractionation on a Superdex 200 column (ÄKTA Start) using PBS as the running buffer. A protein peak was detected at 280 nm, and fractions corresponding to molecular weights from 160 to 90 kDa were collected and combined. The combined fractions were aseptically filtered through a 0.2 μm filter and aliquoted under aseptic conditions.

[0285] The protein concentrations of all three PAM formulations were determined using the MicroBCA™ Protein Assay Kit (ThermoFisher Scientific, catalog number 23235).

[0286] Results: SDS-PAGE analysis of the obtained protein formulations (PEG-PAM 5000 (PEGylated via cysteine), PEG-PAM 10000 (PEGylated via lysine), and X-PAM (XTENated via lysine)) showed that all three formulations shifted to higher molecular weights compared to untreated PAM. Figure 15 Furthermore, the enzyme activity of all formulations was determined as described in Example 3 and compared with the enzyme activity of untreated PAM. Figure 15Compared to untreated enzymes, PEGylation via lysine using PEG-10000 retained 88% of the PAM specific activity. XTENation via lysine using the linker retained 76% of the PAM specific activity compared to untreated PAM, indicating that PEGylation with PEG-10000, similar to PEGylation with PEG-5000 (Example 5), substantially does not interfere with PAM activity. XTENation resulted in a 24% decrease in PAM specific activity compared to untreated PAM; however, this decrease may be partly due to an overestimation of the total PAM concentration in the total protein assay method used (Micro BCA™ Protein Assay Kit), possibly influenced by false positives from the linked XTEN peptide, leading to an underestimation of PAM specific activity. Surprisingly, PEGylation via cysteine ​​residues using PEG-5000 resulted in a significant decrease in PAM specific activity, indicating that this modification method is not suitable for PAM enzymes.

[0287] In short, such as Figure 15 As shown, the results indicate that the activity of PAM PEGylated with PEG 10000 via coupling with free lysine residues was slightly reduced compared to unmodified PAM. PEGylated PAM exhibited a 12% decrease in activity. Similarly, coupling the XTEN peptide with free lysine residues resulted in a 24% decrease in activity. Finally, surprisingly, unlike the results of coupling PEG 5000 with free lysine residues as shown in Example 5, coupling PEG 5000 with cysteine ​​resulted in a significant 87% decrease in the specific activity of PAM.

[0288] SEQ ID NO: 1 - prepro-PAM isoform 1 AS 1-973

[0289] SEQ ID NO: 2 - prepro-PAM isoform 2 AS 1-868

[0290] SEQ ID No: 3 - prepro-PAM isoform 3 AS (amino acids 829-896 of SEQ ID No. 1 are deleted)

[0291] SEQ ID No: 4 - prepro-PAM isoform 4 (amino acids 829-914 deleted from SEQ ID No. 1)

[0292] SEQ ID No: 5 - prepro-PAM isoform 5 (isoform 1 with an amino acid added at position 896)

[0293] SEQ ID No: 6 - prepro-PAM isoform 6 (amino acids 897-914 deleted from SEQ ID No. 1)

[0294] SEQ ID No: 7 - PHM subunit of PAM

[0295] SEQ ID No: 8 - PAL subunit of PAM

[0296] SEQ ID No. 9 - PHM fragment (amino acids 31-377 of PAM SEQ ID No. 1)

[0297] SEQ ID No: 10 - Recombinant human PAM fragment sequence with C-terminal amino acid GS added (amino acids 23-834 of PAM isoform 1 SEQ ID No. 1)

[0298] SEQ ID NO: 11 - PAM isoform 1 of prepro-PAM isoform 1 AS 31-973 (deletion of amino acids 1-30 of SEQ ID No. 1)

[0299] SEQ ID NO: 12 - PAM isoform 2 of prepro-PAM isoform 2 AS 31-868 (amino acids 1-30 of SEQ ID No. 2 are deleted)

[0300] SEQ ID No: 13 - PAM isoform 3 of prepro-PAM isoform 3 AS 31-905 (amino acids 1-30 of SEQ ID No. 3 are deleted)

[0301] SEQ ID No: 14 - PAM isoform 4 of prepro-PAM isoform 4 AS 31-887 (amino acids 1-30 of SEQ ID No. 4 are deleted)

[0302] SEQ ID No: 15 - PAM isoform 5 of prepro-PAM isoform 5 AS 31-973 (SEQ ID No. 5 with deletion of amino acids 1-30)

[0303] SEQ ID No: 16 - PAM isoform 6 of prepro-PAM isoform 6 AS 31-955 (amino acids 1-30 of SEQ ID No. 6 are deleted)

[0304] SEQ ID No: 17 - Sequence of recombinant PAM variant 2 (amino acids 31-387 and 495-817 of SEQ ID No. 1)

[0305] SEQ ID No. 18 - Recombinant PAM AS 31-834 of prepro-PAM isoform 1 with added C-terminal amino acid GS

[0306] SEQ ID No. 19 - XTEN peptide

Claims

1. A modified peptidyl-glycine α-amidyl monooxygenase (PAM) having an increased in vivo half-life compared to unmodified PAM, wherein the enzyme activity of the modified PAM is maintained compared to unmodified PAM.

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

3. The modified PAM according to claim 1 or the method according to claim 2, wherein the PAM is a protein comprising a polypeptide sequence selected from SEQ ID No. 7 to 18 or having at least 85%, particularly at least 90%, more particularly at least 95%, or even more particularly at least 99% sequence homology with it.

4. The modified PAM according to claim 1 or the method according to claim 2, wherein the PAM is a protein comprising a polypeptide sequence selected from SEQ ID No. 7 to 16 or having at least 85%, particularly at least 90%, more particularly at least 95%, or even more particularly at least 99% sequence homology with it.

5. The modified PAM or method according to any one of the preceding claims, wherein the enzymatic activity of the PAM is the α-amidation activity of the PAM.

6. The modified PAM or method according to any one of the preceding claims, wherein the PAM is modified in the following manner: a. Connecting one or more natural or synthetic polymer units, wherein the polymer is selected from the group consisting of: i. PEG, particularly in the range of average molecular weight from 0.2 to 100 kDa, particularly from 1 to 90 kDa, more particularly from 2 to 80 kDa, more particularly from 3 to 70 kDa, more particularly from 4 to 60 kDa, and even more particularly from 5 to 50 kDa; ii. XTEN; iii. Peptide polymers composed of repeating PAS (amino acids proline, alanine, and serine) units, particularly containing 100-200, particularly 120-180, more particularly 130-170, and even more particularly about 150 PAS repeating units; iv. Elastin-like polypeptides composed of repeating units of valine-proline-glycine-x-glycine, wherein x is any amino acid other than proline, and particularly composed of 150, 200, 250, 300, 400, or 500 to 550, 600, 750, 850, 950 or 1000 amino acids. v. HAP polypeptide, with the sequence (Gly4Ser)n, where n is 100 to 200; vi. Gelatin-like fusion protein with the sequence (Gly-XY)n, where X and Y are any amino acids except cysteine, and where n = 60 to 1500; vii. Polysaccharides, especially those selected from the following group:

1. Glucan; 2. Hydroxyethyl polysaccharides; 3. Proheparin; 4. Hyaluronic acid; viii. Polysialic acid; b. Conjugated to serum proteins such as albumin or immunoglobulins or partial conjugations of immunoglobulins; c. Site-directed mutagenesis by inserting, deleting, and / or exchanging one or more amino acids within the amino acid sequence of PAM.

7. The modified PAM or method according to any one of the preceding claims, wherein the in vivo half-life of the modified PAM is increased by at least 2 times, particularly at least 5 times, more particularly at least 10 times, more particularly at least 20 times, more particularly at least 50 times, more particularly at least 100 times, more particularly at least 150 times, more particularly at least 250 times, more particularly at least 350 times, and / or Compared to unmodified PAM, the modified PAM exhibits an in vivo half-life 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 The in vivo half-life of the 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, and more particularly at least 60 hours.

8. The modified PAM or method according to any one of the preceding claims, wherein the PAM level in a sample obtained from a subject who has received at least one dose of the modified PAM is increased compared to a pre-drug sample obtained from the subject before receiving the at least one dose of the modified PAM, wherein the sample is obtained from the 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 the last dose of the modified PAM is administered to the subject.

9. The modified PAM or method according to any one 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 enzyme activity is maintained compared to unmodified PAM.

10. The modified PAM or method according to any one of the preceding claims, wherein the modified PAM, when administered to a subject, increases the bioavailability of one or more amidated peptides, particularly one or more peptide hormones, in the subject.

11. A pharmaceutical composition comprising the modified PAM according to any one of claims 1 to 10.

12. The pharmaceutical composition according to claim 11, wherein the pharmaceutical composition is an ready-to-use formulation.

13. The pharmaceutical composition according to any one of claims 11 to 12, wherein the pharmaceutical composition is a freeze-dried composition.

14. The modified PAM according to any one of claims 1 to 10 or the pharmaceutical composition according to any one of claims 11 to 13, for achieving an increase in PAM level and / or α-amidation activity level in the blood circulation of a subject, particularly an increase of at least 2 times, particularly at least 3 times, particularly at least 5 times, particularly at least 9 times, particularly at least 10 times, 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. The modified PAM according to any one of claims 1 to 10 or the pharmaceutical composition according to any one of claims 11 to 13, used as a medicine, particularly for treating diseases or conditions selected from the group consisting of: cardiovascular, renal, inflammatory, infectious and metabolic diseases or conditions, dementia, cancer and other diseases or conditions associated with peptide homeostasis disorders, wherein more particularly, said diseases or conditions are sepsis.

16. The modified PAM according to any one of claims 1 to 10 or the pharmaceutical composition according to any one of claims 11 to 13, for reducing the risk of a subject contracting a medical condition, and / or for reducing the incidence of a disease or symptom in the subject, and / or for reducing the severity of a disease or symptom in the subject.

17. The modified PAM according to any one of claims 1 to 10 or the pharmaceutical composition according to any one of claims 11 to 13, for use in treating or preventing a disease or medical condition in a subject, wherein the disease or medical condition is associated with endothelial barrier dysfunction and / or blood-brain barrier dysfunction.

18. The modified PAM of claim 17 for treating or preventing a disease in a subject, wherein the disease or medical condition is selected from the group consisting of: cardiovascular, renal, inflammatory, infectious and metabolic diseases or conditions, dementia, cancer and other diseases or medical conditions associated with peptide homeostasis disorders, wherein more particularly, the disease or condition is sepsis.

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